Grants

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.

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.
CD68

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.  

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. 

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 aim to define the subclonal diversity of DIPG with a view to better understanding the evolutionary dynamics underlying this variation. Firstly, we will use high-depth sequencing to explore the subclonal architecture of a series of DIPG specimens. We will generate direct evidence of subclonal diversity by longitudinal studies of biopsy/autopsy pairs for which multiple topographically distinct samples are available post-mortem. We will further explore the functional consequences of this heterogeneity by studying single cell-derived colonies derived from primary tumour specimens in vitro, using advanced high-throughput image analysis linked to targeted resequencing. The long-term goal of such an approach is to provide a framework for preclinical testing of evolutionary biologydriven combinatorial therapies, and to generate data to underpin novel, rationally designed clinical trials in these currently untreatable diseases. 

Brain tumors are the largest group of solid tumors and the leading cause of cancer-related deaths in childhood1. The most devastating of these is DIPG, an incurable brainstem tumor with a median survival of less than one year2,3. DIPGs show poor response to conventional radiation and chemotherapeutic strategies used in adults. Only within the last decade have studies really begun to describe differences between the adult and pediatric disease, underscoring the need for better therapies targeted to the pediatric disease. Most recently, our group reported a major breakthrough in our understanding of DIPG biology with the identification of three molecular subgroups of DIPG4: MYCN, Silent and H 3K27M. The MYCN subgroup has no r ecurrent mutations but is instead characterized by DNA hypermethylation, high grade histology, and chromothripsis on chromosome 2p leading to recurrent high level amplification of MYCN and ID2. The Silent subgroup is characterized by silent genomes with a lower mutation rate than the other two subgroups. The H3K27M subgroup is the largest, and all DIPGs in this subgroup harbor a mutation in either H3F3A or HIST1H3B. In addition, this group is characterized by highly unstable genomes, global DNA hypomethylation and co-occurrence of additional genetic alterations including PDGFRA amplification and ACVR1, PIK3CA and TP53 mutations. Discovery of three molecular subgroups has revolutionized our thinking about the pathogenesis of DIPG6-8 and highlighted the importance of epigenetic dysregulation during tumorigenesis, although it is still unclear which of these alterations represent feasible therapeutic targets. We hypothesize that combined expression of frequent, subgroup-specific, gene alterations in mouse neural stem cells will result in the dysregulation of normal cellular processes, ultimately leading to malignant transformation. In this study we will elucidate the interactions between frequently altered genes in each of the DIPG subgroups and the mechanisms underlying their role in tumorigenesis through two specific aims: 1. Transformation of mouse neural stem cells using lentiviral delivery of genetic alterations associated with each of the three DIPG subgroups.  2. Development of new mouse models of each DIPG subgroup that can be used for pre-clinical drug testing.  Co-expression of subgroup-specific alterations in mouse neural stem cells will allow us to systematically determine the effect of individual alterations on normal cellular processes and their ability to induce malignant transformation and/or maintain the malignant phenotype. Subsequent injection of the mouse neural stem cell lines into the brainstem of immunocompromised mice will enable us to conclude which combination of alterations is both necessary and sufficient for tumor formation and maintenance in vivo. We will then remove the mutant histone to test whether this is important for tumor maintenance in vivo and could thus serve as a therapeutic target.  These experiments will inform production of subgroup-specific transgenic mouse models for DIPG and address a gap in the development of successful DIPG clinical trials. Development of accurate preclinical mouse models of DIPG is crucial to understanding why current therapies fail and for identifying novel drugs that can effectively treat children suffering from this devastating disease. Our group is a leader in the field of DIPG and we are well poised to translate discoveries made through this project into novel and more successful therapeutic trials for this fatal disease.  

Hypothesis- Our hypothesis is that a peptide vaccination targeting H3.3K27M will be an effective tumor-specific therapy for patients with gliomas expressing this mutation, without antigen escape or toxicity. 
 
Goals- To identify an effective therapy that will prolong the survival of children with DIPG. We will pursue the following two specific aims: 
 
1. To design an H3.3K27M peptide vaccine and determine if it is immunogenic. 2. To determine if H3.3K27M peptide vaccine can significantly prolong the survival of H3.3K27M mutant DIPG-bearing mice. 
 
Background- Diffuse intrinsic pontine glioma is a type of brain cancer that arises in children and is incurable. Recently K27M mutations were described in H3.1 and H3.3 in up to 80% of human DIPGs.    
 
Clinical Significance DIPG is an incurable tumor.  We are proposing to explore a new therapeutic strategy by developing a peptide vaccine against the mutant histone, a tumor specific antigen.  Successful completion of this project will provide the scientific rationale for a clinical trial of a peptide vaccine for children with DIPG whose tumors harbor the H3.3K27M mutation (60% of children with DIPG). 
 
Design and Methods of the proposed study- This proposal brings together two labs: the Becher lab and the Sampson lab.  The Becher lab has expertise in genetically engineered mouse models of DIPG and the Sampson lab has expertise with the development of peptide vaccines for gliomas.  Aim 1 will be performed in the Sampson lab and Aim 2 will be performed in the Becher lab. Aim 1- To design an H3.3K27M peptide vaccine and determine if it is immunogenic. We propose to develop and test a vaccine consisting of a peptide encompassing the mutant amino acid- methionine substituting for a lysine.  Our peptide design will be a 25-mer and will be called PEPH3.3K27M. The mutant amino acid is highlighted 15APRKQLATKAARMSAPSTGGVKKPH39.  The control 25-mer peptide will be called PEP-H3.3WT: 15APRKQLATKAARKSAPSTGGVKKPH39.  We will vaccine mice at day 1, 7, 14 and on day 21 and assess the immune response by IFNγ ELISpot to wild type and mutant peptides. 

Aim 2-  To determine if H3.3K27M peptide vaccine can significantly prolong the survival of H3.3K27M mutant DIPG-bearing mice. we propose to test the efficacy of the PEP-H3.3K27M peptide vaccine in mice bearing H3.3K27M mutant DIPGs. Cohorts will receive intradermal PEP-H3.3K27M vaccination or intradermal PEP-H3.3WT vaccination at the tail base once weekly for three weeks starting at 3 weeks post injection of virusproducing cells (postnatal D3-4).   If we do not observe significant efficacy, we will then attempt strategies designed to overcome these barriers such as T-cell immunomodulatory agents (lymphodepletion with temozolomide) and/or adjuvants (KLH). 

Hypothesis: Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an almost universally fatal prognosis. Despite many clinical trials testing numerous chemotherapies and targeted systemic agents, both concurrent with RT and in the adjuvant setting, these therapies have not changed OS. We and others believe that 3 major issues contribute to the poor response to therapy in DIPG patients treated with RT and concurrent systemic therapies. First, drug penetration into DIPG tumors is severely limited by the blood brain barrier. Second, many systemic therapies have been tested in DIPG based on extrapolation from adult high grade glioma trials, without a clear rationale for testing in this genetically distinct tumor type. Finally, the evidence underlying the radiosensitizing potential of many of these agents is lacking. We hypothesize that nanoparticlemediated and convection-enhanced (CED) drug delivery, coupled with the use of targeted radiosensitizers which have been validated in DIPG cells, will lead to higher rates of local control and consequent OS for this disease. As described below, we will test this hypothesis in a proposed one year pilot study focused on the pre-clinical development of novel DIPG radiosensitizers.   Background: As noted above, DIPG is a devastating tumor, with most patients succumbing to their disease within 2 years after diagnosis. Focal RT is the only treatment which has shown a benefit in DIPG, although it extends OS by only ~3-4 months. Remarkably, numerous chemotherapy combinations, RT fractionation regimens, and novel systemic agents have been tested in DIPG, without any appreciable OS improvements in the last 30 years. In addition, many agents were tested in DIPG based on observed activity in adult GBM, and temozolomide (TMZ) is one example. Unfortunately, studies now confirm that TMZ is not effective in DIPG when added to standard RT.  Given that virtually all DIPGs recur locally, there has been great interest in testing novel combinations of targeted systemic therapies with RT, as a means to enhance local control via radiosensitization. Examples include small molecules targeting Aurora Kinase B, VEGFR-2 and PDGFR. DSB repair pathways play critical roles in the response to IR, as highlighted by the exquisite radiosensitivity of cells from patients with inherited DSB repair defects. It is now known that key DNA damage and checkpoint genes are up-regulated in DIPG, including the Chk2 and WEE1 kinases, suggesting a role for DSB repair inhibition as a radiosensitization strategy. While these are promising strategies, recent studies suggest that the efficacy of DIPG therapies is severely limited by drug delivery, which suggests novel methods are needed to ensure adequate tumor targeting. Goals, Design and Methods: We have recently performed a high-throughput screen for novel DNA double-strand break (DSB) repair inhibitors, which led to the identification of several lead compounds with radiosensitizing activity in vitro. We propose to test these compounds as potential radiosensitizers in primary DIPG cells in vitro, and several of the most promising lead hits will then be encapsulated in nanoparticles to facilitate drug delivery into DIPGs in vivo. Finally, three molecules will be selected for testing as radiosensitizers in a DIPG xenograft mouse model in vivo. Clinical Significance: Our proposed studies have the potential to identify novel therapeutics which can be tested in DIPG patients, and they represent an important proof-of-principle, which can be used as the basis to test other drugs as novel DIPG radiosensitizers in the future.   Research Team: This application brings together two new Principal Investigators (PI’s) from Yale Medical School, Bindra and Zhou, with complementary research expertise in DSB repair and nanoparticle drug delivery, respectively. Importantly, they share a common goal of developing novel therapeutics for the treatment of adult and pediatric high grade gliomas. Bindra is a physicianscientist in the Department of Therapeutic Radiology, with a clinical and research focus on pediatric CNS tumors, including DIPGs. His laboratory is developing cell-based screening approaches to identify novel radiosensitizers for high grade gliomas. He is particularly interested in eventually translating these discoveries into the clinical setting, via the development of investigator-initiated Phase I trials. Zhou is a translational scientist in the Department of Neurosurgery, with substantial experience in nanoparticle research and CNS drug delivery. His laboratory is focused on developing novel, nanoparticle-based drug delivery systems. 

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is the leading cause of brain cancer associated deaths in children despite constituting only 10-15% of childhood brain tumours.  Approximately 300 children per year with a median age of 6-7 years will be diagnosed with DIPG in the US [about 20 per year in Australia].  Currently, radiotherapy is the only treatment offered as no effective chemotherapeutic is available and surgery is not an option due to tumor location.  As a result, the median survival time is less than 12 months.  This statistic unfortunately has not changed in over 35 years of investigation and highlights an urgent need for new and novel methods of targeting DIPG.   SCIENTIFIC MERIT AND FEASIBILBITY: Our research program identifies targeted therapeutics, both small molecule inhibitors and monoclonal antibodies, directed to cell-surface receptor tyrosine kinases (RTKs) that have potential efficacy in brain cancer.  We are now beginning to apply this expertise to DIPG. DIPG is a highly heterogeneous tumor with a variety RTKs responsible for tumor initiation (PDGFRα), growth (EGFR), vascularisation (VEGFRs), dissemination (c-Met) and stem/progenitor cell maintenance (c-Kit).  Although a number of clinical trials are under way using small molecule inhibitors and humanised monoclonal antibodies targeting EGFR with nimotuzumab [1], VEGFR and EGFR with Vandetanib [2] and PDGFR with Dasatinib [3], most have had limited success and appear to target one or two oncogenic proteins.  In the first part of our proposal we will use a multi-targeting agent (AMG706 targeting PDGFRs, VEGFRs, c-Kit) and the single targeting agents AMG102 (targeting HGF, the c-Met ligand) and panitumumab, which targets wild type EGFR (EGFRwt) and the truncation mutant EGFRvIII), alone and in combination, to inhibit multiple cellular subpopulations and multiple growth promoting RTKs expressed in DIPG.  In addition, we will investigate the novel anti-tumor activity of human amniotic stem cells (hASC) in DIPG, as these cells have demonstrated antiRTK activity and apoptosis inducing activity in glioblastoma multiforme (GBM) in vitro and in vivo.  Our extensive expertise in brain cancer research and access to novel therapeutic agents makes this proposal scientifically significant and highly feasible.     HYPOTHESES:   1: Targeting multiple activated RTKs in DIPG will be an effective therapeutic strategy;  2: hASC will have anti proliferative and anti-tumorigenic effects against DIPG sphere lines;  3: The combination of both these strategies will have enhanced anti-tumor activity in DIPG. SPECIFIC AIMS  We will determine: 1: the anti-tumor activity of AMG706 in combinations with AMG102 and panitumumab against a panel of patient derived DIPG sphere cell lines in vitro and in vivo by conducting tumorigenicity-related functional assays (e.g. proliferation, cell cycle, apoptosis assays), and assessing RTK expression and activation, and orthotopic xenograft growth;  2: the anti-tumor activity and mechanisms of action of hASC against DIPG sphere cell lines in vitro and in vivo as described in Aim 1; 3: the anti-tumor activity of the therapeutic strategies in Aims 1 and 2 in in vivo subcutaneous and orthotopic xenograft models of DIPG. PROJECT GOALS:  To identify novel anti-tumor therapeutic strategies for DIPG DESIGN AND METHODS:  Our study will utilize primary DIPG sphere cell lines isolated from autopsied material to evaluate new therapies both in vitro and in orthotopic xenograft models of DIPG.   CLINICAL SIGNIFICANCE:  This study will evaluate several targeted therapeutics (some that are in clinical trials and one that is approved by the Food and Drug Administration) that recent discoveries suggest should have anti-tumor activity in DIPG. As these therapeutics are already in the clinic, and because we will evaluate them in clinically relevant DIPG xenograft models, there will be the opportunity to rapidly translate them into the clinic. In this proposal, the therapeutic value of hASC in DIPG will also be assessed. These cells are already being used clinically in our institute; therefore, if our data indicate that they have therapeutic potential, we have the ability to conduct a trial in patients with DIPG. In the long term, we hope to design new therapeutic strategies that extend the lives of patients with DIPG. 

Diffuse Intrinsic Pontine Glioma (DIPG) is a rare high-grade glial tumor that occurs in young children and is nearly uniformly fatal. Several factors have contributed to the high fatality in children with DIPG. It occurs in, a region, making surgery potential dangerous. Complete surgical resection of tumor is impossible because of the highly invasive nature and ill-defined boundaries of the tumor. Radiation has been used in DIPG treatment, but it is not curative and merely extends life by a very short time. Delivery of chemotherapeutic agents to the tumor has been hindered by the existence of the blood brain barrier. Doses of drugs that result in significant systemic toxicity have to be administered to obtain minuscule reduction in tumor growth. Thus, there is a dire need for novel therapeutic approaches to improve survival in children with DIPG.

In the current application, we propose to evaluate the feasibility of using  ex-vivo  expanded  natural  killer (NK) cells to target DIPG tumor cells in pre-clinical studies. NK cells are a subset of  lymphocytes characterized by CD56 positivity and CD3 negativity (CD56+, CD3-). Their use for cancer therapy is particularly appealing because these immune cells unlike T- and B-cells can lyse tumors without prior immune sensitization. NK cell-mediated cytotoxicity is highly dependent on their ability to distinguish "self' (normal cells) from non-self (tumor cells). Our vitro data show that (a) DIPG cells are sensitive to NK-mediated cytolysis and (b) histone deacetylase inhibitors (HDACis) upregulate genes such as NKP30 and NKG2D, which are critical for NK-tumor recognition of tumor cells. Here. we will build upon these findings to assess if loco­ regional administration of NK cells [using a guide screw or convection enhanced delivery (CED)] in mouse orthotopic models of DIPG will promote tumor shrinkage. We will also ask if HDACI-mediated increase in tumor cell recognition by NK cells will augment DIPG cell cytolysis.

The specific aims of our study are:

Aim I. To study the cytolytic activity of NK cells delivered loco-regionally in mouse orthotopic models of DIPG. We will first evaluate the cytolytic activity of ex vivo expanded NK cells from five other anonymous donors using fluorescence-based cytolysis assays. NK-sensitive, firefly luciferase marked DIPG cells will be implanted in the brain stem of mice and treated with graded doses of NK cells labeled with a fluorescent lipophilic dye (DIR) and delivered by a guide screw or convection enhanced delivery (CED). Tumor response will be measured by bioluminescence imaging. NK persistence will be assessed by fluorescence imaging.

Aim II. NK cells will be combined with an HDACI, MS-275, which we previously showed to upregulate tumor recognition molecules on NK cells to evaluate its effect on NK cell cytolytic activity in vitro. In vivo, we will combine the two agents at doses where each alone promotes 20% killing. The involvement of tumor recognition molecules in augmentation of NK function will be shown by using specific blocking antibodies.

Feasibility and Innovation: Feasibility is shown by: (a) the proven expertise of our multi-institutional and multi-disciplinary team in basic and translational neuro-oncology (b) availability of platform technologies that we have developed, using artificial antigen presenting cells (aAPC) to expand clinical-grade NK cells (b) our unique brainstem DIPG mouse model and cell lines generated from autopsy material and finally (c) our expertise in delivering chemotherapy to the brain stem using guide screw and CED.

Clinical Relevance and Significance: The proposed project is based on our unique access to and ability to use clinically-adaptable agents: (i) aAPC, (ii) NK cells and (iii) narrow-spectrum HDACi, Our group has ongoing clinical trials infusing NK cells to treat children with non-neural tumors. Based on our recent pre-clinical research on medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) (section III-C and data not shown), we are in the process of seeking regulatory approval to open the first in pediatrics immunotherapy trial for children with fourth ventricular tumors through loco-regional administration of ex vivo expanded NK cells. The successful completion of this study will (a) provide pre-clinical evidence for the therapeutic application of NK cells in children with DIPG and (b) provide a rationale for novel synergistic combinations of NK cells and other therapeutic agents. The proposed work has the potential to create a paradigm shift in DIPG treatment.

Brain tumors are the largest group of solid tumors and the leading cause of cancer-related deaths in childhood1. The most devastating of these is DIPG, an incurable tumor with a median survival of less than one year2,3.  DIPGs show poor response to conventional therapy and until recently we have lacked knowledge of the molecular profile of DIPG hindering development of targeted therapeutics. Toward a better understanding of the molecular profile of DIPG, our group at the Hospital for Sick Children was the first to institute a post-mortem tissue collection protocol for DIPG patients and to use this material to obtain high resolution molecular genetic profiles of these tumors. Through highresolution DNA and RNA microarray analysis of these tumors we uncovered potential therapeutic targets, including PDGFRA, PARP and aurora kinase B4,5, for which therapeutic agents are already available. We have since expanded our protocol to include centers from across Canada and the U.S. and now have one of the largest DIPG tissue banks, with matching clinical data in the world.  Discovery of recurrent histone mutations in pediatric GBM has revolutionized our thinking about the pathogenesis of DIPG6-8. A highly recurrent mutation was found in variant histone H3.3 at amino acid 27 resulting in lysine to methionine substitution (H3.3K27M). While recent data suggests that the presence of the mutated histone results in global loss of methylation of K27 through inhibition of the histone methyltransferase EZH2, how this causes cancer and whether blocking the effects of this mutation can be used to treat DIPG is still unknown. In Aim 1 we propose an siRNA screening approach to identify drugs targeted specifically to DIPGs carrying the K27M mutation. Our siRNA screen is designed to identify genes that are directly targetable through specific small molecule inhibitors. Despite this huge explosion of genomic data, there has been little movement in identifying which of these mutations, or over-expressed genes in DIPG are bona fide therapeutic targets. Further, we still have little understanding of the reasons for the minimal therapeutic effect of traditional agents, such as radiation and temazolamide, which seem to provide at least some survival benefit for adult patients with high grade astrocytomas. Therefore there is a critical need to understand the basis of this therapeutic resistance in DIPG and translate this into effective treatment protocols for these children.  In adult GBMs, the protein MGMT has been shown to be an important mediator of resistance to temozolamide and patients whose tumors express this protein have a worse overall survival. However, we have demonstrated that MGMT is infrequently expressed in DIPG suggesting an alternate mechanism of therapeutic resistance exists. Another major pathway involved in repairing alkylating-agent mediated damage is the base excision repair pathway. Our data shows that this pathway is highly active in pediatric GBM and that inhibiting this pathway can restore response to temozolamide in both in vitro and in vivo models. We hypothesize that resistance to traditional forms of treatment, namely radiation and alkylating agents such as temozolomide are promoted through the base excision repair pathway in DIPG. The potential for blocking the base excision repair pathway to overcome resistance of DIPGs to radiation and alkylation agents will be tested in Aim 2. The protocols developed in Aim 2 can then be used to test targets from Aim1 in the future. Our approach will yield highly promising drug targets that can be readily tested in DIPG models and rapidly transitioned to phase I clinical trials. We have the facilities and expertise at the Brain tumor research at The Hospital for Sick Children in Toronto that will allow us to take these preclinical model approaches and eventually transition into clinical trials with our dedicated team of neuro-oncologists. Our innovative approaches of targeting novel DNA repair proteins in combination with standard therapy and our functional siRNA screen will allow us to identify novel therapeutic strategies to treat children with DIPG.  

Scientific Merit: The prognosis of diffuse intrinsic pontine gliomas (DIPGs) has not improved significantly over the last 40 years.  A major reason for this limitation is the lack of an understanding of the underlying biology of these tumors, as well as the absence of appropriate targets for therapeutic intervention.  The biologic material for DIPGs that is available consists predominantly of autopsy specimens.  While the analysis of these samples will provide important clues about the disease, the lack of material at the time of diagnosis continues to hamper significant progress.  With dramatic advances in neurosurgical techniques that now permit the safe biopsy within the brainstem, coupled with dramatic advances in molecular profiling of very small quantities of tumor material, we can now move forward towards a direct examination of these tumors prior to the influence of radiation and chemotherapy.  In collaboration with the Dana-Farber/Harvard Cancer Center, the Dana-Farber Center for Cancer Genome Discovery and the Broad Institute, as well as the active participation of approximately 20 of the largest pediatric neuro-oncology programs in the country, we are ready to embark on a new direction in the treatment of DIPG.  
 
Disease Impact: Our approach to DIPG over the last 40 years speaks for itself.  Not only do the innumerable negative clinical trials speak about our failure to change the course of this disease after all of this time, but also, we are no further ahead in terms of our understanding of these tumors or their treatment.  As such, bold steps are needed to change our current approach and this proposal addresses this issue head on. 
 
Innovation: The current clinical trial proposal is innovative in multiple ways.  It builds on our improved neurosurgical expertise, thus allowing us to obtain tissue from newly diagnosed patients with the classical clinical and radiographic criteria of DIPG.  We will then treat patients based on components of the individual analysis of their tumor for expression of EGFR and MGMT.  This will be the first major effort of this kind and will provide important answers to the role of MGMT and EGFR for patients with newly diagnosed DIPG.  While any therapy combination can be criticized and replaced by different combinations, the 4 agents selected for this trial meet the following important principles.  First, radiation therapy is the standard of care and provides important effects on the tumor.  We have also included bevacizumab for all patients based on the expression of VEGF in all malignant gliomas (recall that vascular proliferation is one of the diagnostic criteria of these tumors).  While the presence of vascular proliferation is largely based on autopsy cases in DIPG patients, the rational for this approach is justified in newly diagnosed patients as well.  The use of temozolomide is only appropriate in the absence of MGMT expression and this protocol will avoid this agent in patients unlikely to benefit, while allowing those that are MGMT negative (even if this is a small subset of patients) the opportunity to receive the drug and evaluate its potential activity.  Finally, EGFR has been demonstrated in a number of DIPG autopsy cases, and this protocol will allow us to settle the relevance of this pathway in these tumors.  Most importantly, all samples will undergo extensive molecular profiling to identify new pathways and targets for potential intervention in future studies. 
 
Feasibility: The ability of the French group at Institut Gustave Roussy to complete more than 120 consecutive biopsies without mortality or prolonged morbidity demonstrates that biopsy of the pons can be performed.  We have now performed 15 biopsies through this effort without significant morbidity and no mortality.  We have already demonstrated the validity of all of the molecular techniques proposed in this protocol using samples from pediatric low-grade gliomas.   
 
Expertise: We have been able to bring together approximately 20 major pediatric neurosurgical programs in the United States to obtain samples, which can then undergo molecular profiling through the Dana-Farber/Harvard Cancer Center, Dana-Farber Center for Cancer Genome Discovery and the Broad Institute.   We have clinical pediatric oncologists with experience in the administration of the agents being proposed and have included significant oversight to ensure that all patients receive the same high standard of care. 

The objective of this application is to examine if combining ionizing radiation with an oncolytic virus SVV-001 would lead to synergistically enhanced tumor cell killing and significantly improve therapeutic efficacy in vivo in patient tumor-derived intra-brainstem xenograft 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. Traditional challenges for the development of new therapies include the lack of clinically relevant animal model systems, difficulties of drug delivery across the blood brain barrier (BBB) and limited options of targeting therapy-resistant tumor cells. Fortunately, we have established a panel of nine orthotopic xenograft mouse models of DIPG in the brain stems of SCID mice. Thanks to the generous support of the Cure Starts Now Foundation, we have completed a research project entitled “Eliminating therapy resistant DIPGs with oncolytic picornavirus SVV-001” and identified Seneca Valley Virus (SVV-001) as an attractive agent for DIPGs. Our data confirmed that SVV-001 can infect and effectively kill DIPG tumor cells in vitro, and pass through the BBB in vivo, leading to improved animal survival in a subset of intra-brain stem DIPG xenograft models. However, when compared with greater than 80% cell killing in vitro, the animal survival time was only prolonged~10% (P <0.05), indicating the limited efficiency of SVV-001 as a single agent.
To address such response discrepancies between the i nvitro and in vivo activities and to see knew strategies to further improve therapeutic efficacy, we examined the cellular content of mitochondria, which is the recognized assembly station of many viruses 1-6. We found that mitochondria were very rich in the cultured DIPG cells, but scarce in many xenograft tumor cells derived from the same models. This has led us to hypothesize that the lack of mitochondria in the xenograft tumor cells in vivo impaired the intracellular replication of SVV-001 and subsequently weakened the oncolytic cell killing of the target tumor cells. Fortunately, we have found that ionizing radiation can activate robust mitochondrial biogenesis in vivo, converting mitochondrial-depleted tumor cells to mitochondria-rich tumor cells. Indeed, our preliminary studies involving pediatric GBM have shown that combining fractionated radiation (2 Gy/day for 5 days) with single i.v. injection of SVV-001 led to significant improvement of animal survival times. Since many of the DIPG tumor cells were shown to be responsive to SVV-001 in vitro (they may therefore be inherently permissive to SVV-001), it is therefore our second hypothesis that radiation induced increase of mitochondria would provide SVV-001 the much needed replication “facilities”, thereby converting the “dormant” tumor cells into responsive tumor cells that can be killed. To test these hypotheses, we will utilize our established orthotopic xenograft mouse models to accomplish the following Specific Aims: 1) To prove that lack of mitochondria causes SVV001 resistance in the permissive DIPG tumor cells in vivo; 2) To demonstrate that ionizing radiation activates mitochondrial biogenesis in previously mitochondria-deficient/depleted tumor cells and sensitizes them to SVV-001 induced cell death; 3) To demonstrate that combining ionizing radiation with intravenously injected SVV-001 can improve therapeutic efficacy in vivo, leading to significantly improved animal survival.
The innovations of our proposed studies are two fold. First, it lies in our novel use of a relatively large panel of intra-brain stem DIPG xenograft mouse models. These models were derived from terminal stage DIPG patients and represent the clinically proven therapy-resistant DIPGs that are in desperate need of new therapies. Our models have provided us with unprecedented opportunities to study the biology and to test new therapies in vivo in a micro environment that is the closest to human DIPGs. Secondly, it lies in our prospective examination of the therapeutic efficacy of a novel combined treatment that is rationally designed based on the molecular mechanisms that are complementary to each other. SVV-001 has many features that make it particularly attractive for brain tumors. Identifying the underlying mechanism of SVV-001 resistance and the subsequent development of a new strategy to overcome such resistance would prevent the premature drop-out of a potentially effective therapy. More importantly, since our approach is built on the top of radiotherapy, the standard treatment for DIPGs, our chances of rapid translation into clinical trials are therefore greatly improved.

Scientific Merit: Pediatric diffuse intrinsic pontine gliomas (DIPGs) are high-grade cancers of the brainstem that are rarely biopsied due to the morbidity of the procedure.  Recent advances in the understanding of DIPG biology include the discovery of a mutation in the gene coding for histone 3.3 (H3.3K27M).  However, the exact molecular mechanism of the H3 mutation that leads to tumor formation is not understood.   
 
One of the major obstacles in understanding the biology of DIPGs is the lack of tissue for molecular studies.  In this proposal, we aim to share resources (specimens, data, and expertise) between the Children’s Research Institute (CRI) in Washington DC, and the Hospital for Sick Children (SickKids) in Toronto, Canada to study the transcriptome and proteome of DIPGs.  Two Aims are proposed: AIM1. To map protein expression patterns of DIPG subtypes based on H3 K27 mutation status and activation of SHH/Myc pathways using our DIPG Protein Atlas and SILAC-based targeted proteomics. AIM2.  To generate comprehensive transcriptomic data using RNA-seq for our DIPG cohort and integrate with matching proteomic data. 
 
Disease Impact: We propose to generate targeted proteomics and RNA sequencing of DIPG specimens.  The immediate outcomes of the proposed project will be: i) the identification of genomic mutations, deletion, amplification and fusion.  We will further validate fusion proteins by comparative analysis of proteomics and mRNA data ii) identification of DIPG subtypes based on their histone 3 mutation and further mapping of SHH and Myc pathways in DIPG subtypes.  Generation and comparative analysis of DIPG transcriptome and proteome will have immediate impact on disease by providing new avenues for developing targeted treatments.     
 
Innovation: The proposed project will be the first study to investigate targeted proteome and transcriptome of DIPGs.  CRI has generated preliminary data indicating molecular subtypes of DIPGs as well as their molecular subtypes with regards to histone 3 mutations.  This proposal will use a novel Human DIPG Proteome Atlas to perform targeted proteomics, where proteins involved in each pathway will be mapped using mass spectrometry technique.  Moreover, because DIPG samples will be spiked with DIPG Proteome Atlas, we will be able to generate absolute quantification of expressed proteins.  Furthermore, RNA-seq will generate the opportunity to discover genomic deletion, mutation and amplification.  Comparative analysis with proteome data will enable us to detect expressed fusion proteins in DIPGs and explore their role in tumor formation using targeted proteomics.  As such, this is a highly innovative proposal that will foster collaboration and expand our understanding of DIPG biology.  

Feasibility: CRI and SickKids have shared all frozen specimens and both institutions are capable of carrying the planned experiments as outlined in the proposal.  CRI has generated protein profiling of DIPGs and has identified pathways for further analysis using targeted proteomics.  SickKids is well equipped with carrying the RNA sequencing Aim.  As such, the proposed project is highly feasible and we predict no difficulties with performing the proposed Aims.  
 
Expertise:  Dr. Nazarian (PI) at Children’s Research Institute has led efforts in studying the protein profiling of DIPGs.  CRI has a state-of-the art Proteomic Core housing a recently purchased QExactive mass spectrometer capable of performing high resolution quantitative and targeted proteomics.  CRI has also generated preliminary proteomics data and has identified target pathways which will be further investigated using targeted proteomics.  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 profile of pediatric brain tumors.  This center houses an Illumina HISeq 2500 platform which will be used for the proposed study.

Diffuse pontine glioma (DIPG) is a devastating pediatric malignancy with very poor prognosis. Virtually all patients with DIPG will die from their disease within1-2 years of diagnosis. Recent attempts to improve survival have been unsuccessful, and currently radiation is the only therapy offering limited benefit. One of the challenges in studying DIPG has been the lack of relevant animal models. The goal of this project is to evaluate a novel therapy in the treatment of DIPG by examining the anti-tumor activities of a series of BMI-1 inhibitors that can pass through the blood brain barriers in a panel of orthotopic xenograft models that have been established from autopsied DIPG materials. (from the lab of Xiao-Nan Li, MD, PhD). We hypothesize that i) Inhibiting BMI-1, a transcription repressor that plays an important role in stem cell renewal, with a series of novel small molecule inhibitors, will result in decreased cell proliferation both in vitro and in vivo in orthotopic xenograft mouse models of malignant pediatric brain tumors that over-express BMI-1. ii) Systemic analysis of known BMI-1 down stream genes/signaling pathways together with global gene expression will facilitate the understanding of mechanisms of action of these novel BMI-1 inhibitors. The evaluation of this series of small molecule inhibitors, developed by PTC therapeutics, will provide the pre-clinical rationale for clinical trials in the future. We will test this hypothesis with the following specific aims:
1. To determine if the BMI-1 inhibitor can kill primary cultured xenograft tumor cells, including those that express putative brain tumor stem cell markers (CD133) in vitro;
2. To determine if oral administration of the BMI-1 inhibitor can eliminate pre-established orthotopic xenograft tumors, leading to significantly prolonged animal survival times; and
3. To elucidate the mechanisms of BMI-1 inhibitors by examining the down stream targets of the BMI-1 pathway and whole genome gene expression profiling.
This proposed study would be carried out utilizing models derived from patient autopsy tissue, which has been transplanted into the brainstem of SCID mice. These models represent a unique preclinical testing platform, in that they resemble the pathology and the microenvironment of the original patient tumors. Initially, xenograft cells will be exposed to this novel agent in vitro and screened to determine the most responsive models, as well as the most effective compound. Both non-stem cells and cells which expressive putative stem cell markers will be evaluated, as BMI-1 is known to play a role in stem cell renewal. Subsequently, we will evaluate the efficacy of treating pre-established orthotopic xenograft tumors in vivo with the most effective compound. Finally, we will try to elucidate the method of BMI-1 induced killing by evaluating downstream targets of BMI-1 after treatment with this new agent.
This is the first preclinical study specifically designed to target therapy-resistant childhood DIPGs in vivo in clinically relevant animal models with a small molecule inhibitor for BMI-1. The utilization of this panel of novel patient tumor-derived orthotopic xenograft mouse models of DIPG, and the prospective examination of the therapeutic efficacy of a novel small molecule inhibitor of BMI-1 makes this an innovative approach. All the reagents and assays are well developed in our laboratory and we are uniquely positioned to accomplish the proposed study. The completion of our proposed study should provide strong preclinical rationale for the initiation of clinical trials in pediatric DIPGs in very near future.