The DIPG / DMG Collaborative has funded $13,890,091 in DIPG/DMG research.
Interested in applying for a grant from the DIPG / DMG Collaborative? Learn more.
Gustave Roussy Cancer Center - $93,071
Ultrasound-induced delivery of Panobinostat in preclinical models
Brain tumors are the leading cause of mortality and morbidity from cancer in children and young adults. Malignant gliomas, including supra-tentorial high grade gliomas (HGG) and Diffuse Intrinsic Pontine Gliomas (DIPG), remain a major therapeutic challenge in pediatric oncology because they are still incurable. The failure of current treatments is explained by the difficulties to do a complete surgical resection, and by the low penetration of drugs commonly used neuro-oncology. This low penetration is related to the existence of the blood-brain barrier (BBB), that consists partly of endothelial cells intimately linked together by tight junctions and prevents about 98% of small molecules and 100% of large molecules to reach the brain parenchyma.
It is now proven that the application on brain parenchyma of low power pulsed ultrasound (US) in combination with an US microbubble contrast agent (UCA) can safely and transiently open the BBB (Hynynen et al, Radiology 2001). A significant increase in penetration of several antineoplastic drugs, high molecular weight antibodies, DNA plasmid and neural stem cells has been observed after US-induced opening of the BBB. Studies on murine models of gliomas even showed an improved tumor control and increasing animal survival. These studies, initially conducted on small animals, have recently been applied by us in primates with confirmation of previous results (Horodyckid et al, J Neurosurg in press).
The skull represents the main obstacle to the use of US in neuro-oncology, since the bone induces distortions and attenuation of ultrasonic energy. Considering the fact that a surgical procedure is systematically carried out during the treatment of primary brain tumors (resection or biopsy), the research team CarThéra™, directed by Pr Alexandre Carpentier, has recently developed an implantable ultrasonic transducer. The concept was described in a recent publication with opening of the BBB in rabbits (Beccaria et al, J Neurosurg 2013). The team was able to demonstrate a significant increase in intracerebral concentration of Temozolomide and Irinotecan in rabbits following US9induced opening of the BBB (Beccaria et al, J Neurosurg 2015). The first clinical trial is under way in La Pitié Salpétrière Hopistal (Paris, France) under the coordination of Pr A. Carpentier (NCT02253212 ; see also Carpentier et al, Sci Transl Med in press).
A few of molecules have shown a very good anti-tumor efficacy on DIPG primary cell lines but many of them have poor diffusion across the BBB (Grasso et al, Nat Med 2015). One of the most effective drugs, the panHDAC inhibitor Panobinostat has recently shown a strong anti-tumor activity in vitro and in vivo, but this molecule presents a low penetrance through the blood-brain barrier (~10% in mouse), and its therapeutic index is limited by hematotoxicity.
There is therefore a strong rationale to explore panobinostat with unfocused US in a preclinical xenograft model of DIPG deriving from treatment-naive biopsies. After adaptation and validation of US parameters in mice, we will explore the pharmacokinetics of panobinostat in blood and brain (pons, cortex, thalamus) in healthy mice and then in DIPG mouse models after opening of the BBB with US. Depending on the results, we will evaluate the anti9tumor activity of panobinostat combined or not with unfocused US in comparison to panobinostat alone in the preclinical xenograft DIPG models. This project has been validated by the ethical committee CEEA 26 and Research Ministry (approval number 2015071316539600 v3, APAFIS#1141). In case preclinical data and safety data in the brainstem are encouraging in mice, we will test the safety of US-mediated BBB disruption in the brainstem of primates. The goal is to obtain the necessary preclinical data to justify the development of a phase I clinical trial to evaluate the feasibility and safety of the technique in children treated for a HGG and DIPG.
The Hospital for Sick Children - $194,260
Exploring the interactome of H3K27M to find therapeutic targets for DIPG
We now have a wealth of genetic and epigenetic information about DIPG. However, relatively little is known about the molecular consequences downstream from H3K27M mutations and how they can lead to cancer formation. It is known that, as well as DNA hypomethylation, H3K27M mutation leads to a global loss of the repressive chromatin mark H3K27me3. H3.3K27M has also been shown to interact more strongly than wild type (WT) H3.3 with the polycomb repressive 2 (PRC2) complex members EZH2 and SUZ12. However, beyond these observations relatively little is known about how H3K27M mutations might alter the histone code through changes in interactions with writers, erasers and readers. This is a critical step in understanding how the H3K27M mutation might drive DIPG pathogenesis and how to develop appropriate therapies for H3K27M mutant tumors. To better our understanding of the effects of these H3K27M mutations, we are undertaking a novel approach to the problem by investigating the protein interactions that are altered by H3.3K27M and H3.1K27M. Histone binding partners whose histone affinity may change in the presence of H3K27M are likely to play a significant role in mediating this mutation’s tumorigenicity. As well as extending our molecular understanding of DIPG, focussing on these interactors offers an attractive new avenue in the search for an effective therapy for this fatal disease. The structural and post-translational plasticity of histone tails means directly targeting H3K27M itself with small molecules is unlikely to be effective. However, understanding how this mutation alters the histone interactome offers a unique opportunity to specifically modulate the activity of those proteins immediately downstream from H3K27M. The goal of this project is to exploit novel proteomics technology (Bio ID) to gain new insight into the functional consequences of the H3K27M mutation. We hypothesize that the H3K27M mutation leads to differential interaction with and function of multiple chromatin modifiers and nuclear proteins beyond PRC2. This hypothesis is supported by our preliminary data and will be rigorously tested through the following specific aims: Aim 1: To determine the differential protein interactions imparted by the K27M mutation on both H3.1 and H3.3 Aim 2: To characterize the H3K27M interaction landscape in DIPG model systems Our project is the first to directly address the question of how the H3K27M mutation in both H3.1 and H3.3 impacts on the H3 protein-protein interaction landscape. A deep understanding how the protein landscape of DIPG changes will not only shed further light on the molecular basis of this disease, but it will also open a new approach for developing more effective treatment regimens for this universally fatal disease. The facilities and expertise at The Brain Tumor Research Centre and the Hospital for Sick Children as well as our close collaborations within the DIPG Registry Group will help us rapidly advance our findings through pre-clinical investigations and eventually into clinical trials.
Cold Spring Harbor Laboratory - $100,000
Allele-specific antisense therapy for DIPG
Diffuse intrinsic pontine glioma (DIPG) mainly affects very young children and has the highest mortality of all pediatric solid tumors. The median survival is only 9 months, with most patients dying within 2 years after the initial diagnosis. The clinical management of DIPG is a major challenge in pediatric neuro-oncology. The tumor's location in the brainstem precludes surgical resection, and systemic or local chemotherapy is not effective for DIPG. The standard of care is focal radiation therapy, which only transiently relieves symptoms and delays tumor progression.
Advances in tumor biology and diagnosis, including genomic analysis of tumors, have resulted in the identification of specific mutations, which provide an opportunity for a targeted approach to DIPG. Unlike many adult brain tumors, DIPG is associated with a specific mutation that is present in greater than 80% of tumor samples, and results in replacement of lysine 27 with methionine in variants of the histone H3 protein. The majority of the mutations occur in the H3F3A gene, which codes for histone H3.3. K27 normally undergoes post-translational modifications that result in a dynamic state of un-, mono-, di-, and tri-methylation, or in acetylation, each of which is associated with distinct epigenetic control, in conjunction with other modifications within the histone tails. The K27 mutation represents a toxic gain of function, resulting in sequestration of the EZH2 subunit of the Polycomb repressive complex 2 (PRC2), the enzyme that catalyzes trimethylation of K27 to silence genes. Thus, K27M is a dominant-negative mutation, which occurs somatically, in a heterozygous state in the tumor cells.
The goal of this study is to selectively knock down the expression of the mutant allele of H3F3A, by means of antisense technology. Antisense oligonucleotides (ASOs) are precision drugs for targeted therapy, which have already been or are being developed for a variety of indications. One highly promising ASO, nusinersen, is well into phase-3 clinical trials, and is being safely and effectively administered to the CNS by lumbar puncture 2-3 times a year, in infants and children with spinal muscular atrophy. The Pl was an inventor of nusinersen. We propose to develop ASOs to precisely and selectively elicit destruction of the mRNA coding for mutant H3.3 K27M, such that the normal H3.3 encoded by the wild-type allele can carry out its normal functions. We expect that treatment with such an ASO will lead to tumor regression or arrest, either by itself, or in combination with other drugs, including small molecules or biologicals.
Funds are requested ($100,000 for 1 year) to establish the feasibility of this antisense approach in vitro. ASOs will be systematically designed, screened, and optimized for their ability to selectively reduce expression of H3F3A K27M mutant mRNA and protein in three independent patient-derived DIPG tumor cell lines. Effects on expression of downstream targets, as well as on cell proliferation and survival, will be measured. The lead ASO(s) should be suitable for future IND-enabling, in vivo pre-clinical studies employing orthotopic-xenograft or genetic mouse models of DIPG. The basic approach could also be extended to other dominant-negative mutations in H3F3A or in other genes causally associated with DIPG or with other gliomas.
Children's Cancer Institute - $143,884
Radiosensitisation of Diffuse Intrinsic Pontine Gliomas by modulating the glucose metabolism
Diffuse Intrinsic Pontine Glioma (DIPG) is the most aggressive of all childhood cancers and the leading cause of brain tumour-related death in children. It accounts for 80% of all brainstem gliomas in children, with about 20-30 new diagnoses in Australia and 200-300 cases in the United States each year. There is no effective treatment for DIPG, and all patients die from this disease. To date, radiotherapy (RT) is the only form of treatment that offers a transient benefit in DIPG. Following completion of RT, almost all DIPGs recur locally within 12 months. Therefore, the identification of therapeutic targets that modulate the radioresistance of DIPG cells offers a pathway to the development of effective therapies. It is well established that derangements in glucose metabolism of cancer cells can lead to radioresistance. Dichloroacetate (DCA), currently being used to treat lactic acidosis, can modify tumour metabolism by activating mitochondrial activity to force glycolysis into oxidative phosphorylation. Our preliminary data have shown that DCA inhibits cell proliferation and has profound anti-tumour activity across a panel of DIPG neurospheres. We have also observed the efficacy of RT is significantly improved when DCA is added. Strikingly, the anti-tumour effect of DCA is further enhanced by the addition of metformin, a widely used anti-diabetic biguanide with anti-cancer activity. This ultimately leads to a more potent radiosensitising effect when triple combination is administered. These findings indicate that targeting glucose metabolism represents a novel radiosensitising approach for incurable DIPG and our objective is to develop effective combination strategies optimal for clinical translation. Our previous work has demonstrated for the first time that DCA successfully sensitises glioblastoma (GBM) cells to RT both in vitro and in vivo. Compared to these GBM cells, DIPG cells are even more sensitive to the treatment of RT/DCA combination, which further warrants prioritisation of testing this combinatorial therapy on DIPG models. The synergy demonstrated with RT, the only standard therapy for DIPG, holds great promise for clinical translation. DCA has been used as an orphan drug for various acquired and congenital disorders of mitochondrial metabolism for decades. It has recently been demonstrated to be an active and well-tolerated in patients with recurrent malignant gliomas in a recent phase I clinical trial. It is also worth noting that human toxicity from chronic DCA exposure is influenced by subject age, and oral administration of DCA is far better tolerated in young children than adults. These findings strongly suggest this that DCA warrants investigation as a radiosensitiser against malignant brain tumours in children. More importantly, our data demonstrated that the anti-tumour effect of DCA can be further enhanced by the addition of other clinically available drugs, thereby maximising the cell-killing effect of RT. The comprehensive research project described here will provide the quantum of data needed to identify the mechanisms underlying radioresistance of DIPG and strategies to overcome them. Given the results generated from this project will be easily and rapidly translated to a clinical trial, the anticipated impact of this research project is of great significance: it may lead to a change in treatment regime resulting in longer survival rates for the paediatric patients with newly diagnosed DIPG. 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. We have established 10 neurosphere-forming DIPG cells as well as 2 orthotopic mouse models of DIPG. PI Shen is an early career post-doctoral researcher with particular expertise in DIPG cell culture and xenograft models, cancer cell biology, drug discovery, radiation oncology. Co-investigator Ziegler is an internationally respected paediatric oncologist leading the translational group at Children’s Cancer Institute (CCI) and Sydney Children’s Hospital (SCH) with the focus of translating laboratory studies directly to the clinic for implementation in early phase clinical trials. 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. The total grant amount of USD $143,884 is requested for this proposed project.
Dana-Farber Cancer Institute - $100,000
Characterizing resistance mechanisms to radiation therapy and adjuvant chemotherapy in Diffuse Intrinsic Pontine Glioma
Children diagnosed with Diffuse Intrinsic Pontine Gliomas (DIPGs) face a dismal prognosis, with rampant progression after initial therapy with chemoradiation and a median survival of less than two years. These tumors are universally fatal, with tumors rapidly exhibiting resistance to the current treatments. Therapeutic strategies that delay or ablate the development of resistance are required to improve survival. In this project we will test the central hypothesis that DIPGs exhibit genomic evolution in response to chemoradiation therapy and that differences in the somatic genetic profiles of DIPGs point to mechanism by which DIPGs acquire resistance. Targeting these resistance drivers could represent novel therapeutic approaches to attenuate the acquisition of resistance. We will also test the hypothesis that over-expression of specific genes contribute to the development of resistance, and that identification of these genes will also represent potential therapeutic strategies to ablate or delay the development of resistance. The goal of this proposal is to characterize the mechanisms through which DIPGs can acquire resistance to standard chemoradiation therapy, which will guide the development of therapeutic strategies to overcome such resistance.
Resistance of high-grade pediatric glial neoplasms such as DIPGs to current therapeutic approaches results in very poor long-term survival. This project will systematically examine such resistance mechanisms specifically in the setting of highly lethal DIPGs. We will address at least two mechanisms through which DIPGs can acquire resistance. First we will profile the somatic genomic landscape of resistant DIPG samples to identify driver resistance alterations. Second we will identify specific genes and pathways which when over-expressed contribute to resistance. The results of this proposal will bear clinical relevance in guiding the development of therapies to overcome resistance. To achieve our aims, we will collaborate with the DIPG-BATs clinical trial to access newly diagnosed and autopsy DIPG samples. We will apply multiple innovative technologies including novel analytical methodologies developed by the Beroukhim laboratory and collaborators to examine genomic heterogeneity and to detect sub-clonal populations, and the utilization of a genome-wide lentiviral ORF library to detect genes and pathways that are likely to contribute to the development of resistance.
An Assistant Professor in Medicine, and attending Neuro-Oncologist, Dr. Rameen Beroukhim has substantial expertise to lead this study. Dr. Beroukhim is an international expert of cancer genomics, in particular of copy-number variations in cancers. He has developed computational methodologies to evaluate somatic genetic alterations from high-throughput datasets, and has considerable experience in studying cancer genomes from data generated by whole genome and whole exome sequencing, with an interest in studying cancer heterogeneity and evolution. His laboratory also hasol significant expertise in perform large-scale pooled lentiviral modifier screens in models of pediatric brain tumors. In addition, his appointments at the Broad Institute and the Dana-Farber Cancer Institute ensure access to a rich and collaborative research environment. Dr. Beroukhim will work in close collaboration with Dr. Keith Ligon and Dr. Mark Kieran to complete this protocol. Both Dr. Ligon and Dr. Kieran have led the DIPG-BATs clinical collaboration and have significant expertise in DIPG.
In summary, this proposal will evaluate evolution of DIPGs in response to chemoradiation therapy. The project will determine genomic and transcriptomic factors that influence the acquisition of resistance. The results will guide strategies to optimize the efficacy of current treatments for DIPGs to improve the survival of children diagnosed with this deadly disease.
Baylor College of Medicine/Texas Children's Hospital - $200,000
Targeting DIPGs through Super-activation of Steroid Receptor Co-activators: An in vivo Study in Patient-derived Intra-brain Stem Xenograft Mouse Models
The objective of this application is to determine if combining ionizing radiation with the newly-described steroid receptor co-activator (SRC) small molecule stimulator MCB-613 will lead to enhanced tumor cell killing and significantly improved therapeutic efficacyin vivo in patient tumor-derived orthotopic xenograft (PDOX) mouse models of diffuse intrinsic pontine glioma (DIPG). DIPG is the most lethal childhood cancer, and virtually all children with this disease die within 1-2 years of diagnosis. Challenges for the development of new therapies include the lack of clinically-relevant and biologically-accurate animal model systems, difficulties of drug delivery into the pons across the blood brain barrier (BBB), and rapid development of drug resistance. Fortunately, solutions to overcome these challenges have emerged: 1) We have established a panel of PDOX models of DIPG in the brain stems of SCID mice. We have successfully utilized these models for pre-clinical in vitro and in vivo studies evaluating the efficacy of new therapeutic agents against DIPG. 2) Colleagues in our institution have recently described a new anti-neoplastic compound—MCB-613—with a novel mechanism of action. Unlike traditional anti-cancer drugs which are aimed to inhibit over-expressed oncogenes/pathways, MCB-613 acts by stimulating a specific series of oncogenic genes, the steroid receptor co-activator proteins (SRCs). SRCs are typically over-expressed in cancer cells, leading to accelerate tumor growth, invasion, and metastatic spread. Counter-intuitively, over-stimulation of SRCs by MCB-613 leads to tumor cells death secondary to increased cellular stress and the generation of toxic reactive oxygen species. The drug has only minimal biological disruption of normal cell homeostasis and shows pre-clinical activity in various cancer pathologies, including invitro efficacy against pediatric DIPGs. 3)Most relevant to the treatment of DIPG, MCB613 not only penetrates the intact BBB but actually accumulates over time in brain tissue.
Our central hypothesis is that treatment of DIPG with MCB-613 in combination with ionizing radiation will prolong animal survival times compared to mice treated with placebo and with radiation alone To test our hypothesis, we will utilize our established PDOX models of DIPG to accomplish the following Specific Aims: 1) Determine if the combination of ionizing radiation with i.p. injected MCB-613 will prolong animal survival compared to placebo and radiation-only treated animals, 2) Understand mechanisms of MCB-613-induced DIPG cell killing, and 3) Identify the cellular and molecular causes of MCB-613 therapy resistance.
This proposal is innovative on several levels. Firstly, our panel of intra-brain stem DIPG PDOX mouse models is derived from terminal-stage DIPG patients; consequently, they represent therapy-resistant disease, which is in desperate need of new therapies. These models provide us with unprecedented opportunities to study tumor biology and test new therapies in vivo in a micro-environment closest to human DIPGs. Secondly, our research proposal is the evaluating the therapeutic efficacy of MCB-613, a newly-synthesized anti-neoplastic compound with a novel mechanism of action. This drug crosses the BBB and accumulates in brain tissue, thus allowing MCB-613 to research the tumor site. Indeed, MCB-613 has already shown promising anti-tumor activities against DIPG cells both in vitro and in vivo.
Completion of our proposed study should provide strong preclinical evidences for the initiation of clinical trials of MCB-613 in children with DIPG. Since our treatment approach combines MCB-613 with radiotherapy, the standard treatment for DIPGs, and our chances of rapid translation of the drug into clinical trials are greatly improved. Additionally, this study will also provide critical insight about the use of SRCs as predicative marker of MCB-613 responsiveness, which in turn should facilitate patient stratification in future clinical applications. Finally, our analysis of drug resistance mechanisms of MCB-613 in vivo in DIPG xenograft tumors should provide new clues for new drug combinations to further improve therapeutic efficacy of MCB-613.
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.