Grants

Background:  Due to their vital location, diffuse intrinsic pontine gliomas (DIPGs) are not treated surgically, and the lack of tumor material for research has greatly limited research efforts [1]. Recently, our group and others have used autopsy tissue, and rare biopsy tissues, for genetic analyses of DIPG to identify the molecular defects that cause this disease [2-5].  In addition to identifying genetic alterations that are similar to those found in adult glioblastomas and other types of cancer, including amplifications and mutations in receptor tyrosine kinases and components of the cell cycle regulatory machinery, a unique recurrent mutation in lysine 27 of histone H3 was identified in 78% of DIPGs [6, 7].  Importantly,recurrent mutations in this gene are seen in 36% of childhood non-brainstem tumors, but are very rare or not detected in adult glioblastomas or in 252 other pediatric tumors [6, 8].  
The hypothesis of our proposal is that histone H3 mutation confers an extremely powerful selective advantage in tumorigenesis, only in the specific context of high-grade glioma in the developing brain, with a particularly important role in developing brainstem. Therefore, experiments to understand the mechanisms through which histone H3 contributes to tumorigenesis must be conducted in a relevant cell background and physiological setting.  Because the specific cells of origin for DIPG are unknown, the most reliable way to ensure that we are working with cells relevant to DIPG is to establish xenografts and cell lines from primary DIPG tumors. 
The goals of our proposal are to establish a renewable resource of primary DIPG tissues that will provide relevant model systems for high-throughput screening to identify compounds that have therapeutic efficacy against DIPG, and for basic research studies to elucidate the mechanisms through which histone H3 contributes to DIPG formation and growth.  To ensure that these models are an accurate reflection of DIPG, and to determine the spectrum of heterogeneity represented by the models, we will perform a detailed analysis of the genomic and gene expression signatures. 
Clinical significance:  Although histone H3 mutations are a unifying molecular defect underlying DIPG, these tumors, like other high-grade gliomas, are heterogeneous in the spectrum of other mutations that they carry, which will likely influence their response to therapeutic intervention.  It is critical to establish a collection of physiologically relevant DIPG models that reflect the heterogeneity that will be encountered in treatment of human DIPGs.  
Design and Methods: 
In Aim 1, we will establish xenografts of DIPGs obtained from autopsies, and serially passage these xenografts to generate a renewable resource of DIPG tissue for research. We will also establish primary cell lines for in vitro experimentation. 
In Aim 2, we will use exome sequencing and gene expression arrays or RNA-seq for detailed characterization of the genetic abnormalities and gene expression signatures in each model established.  We will carefully compare xenografts and primary cell lines to the primary tumor tissue to establish the relevance of the models.  

Diffuse intrinsic pontine glioma (DIPG) is the most lethal childhood cancer, and virtually all children with this disease die within 1-2 years of diagnosis. Major challenges for the development of new therapies include the lack of clinically relevant animal model system,difficulties of efficient drug delivery across the blood brain barrier (BBB) and limited options of targeting therapy-resistant tumor cells (cancer stem cells?). Fortunately, we have established a panel of five orthotopic xenograft mouse models of DIPG in the brainstems of SCID mice, and identified a novel oncolytic virus, the Seneca Valley Virus (SVV-001), that can pass through the BBB and effectively kill brain tumor cells (including brain tumor stem cells). The objective of this application is therefore to examine the invivo anti-tumor activities of SVV-001 in the five intra-brainstem DIPG xenograft mouse models to establish preclinical rationale for the initiation of clinical trials in the near future.
Our hypotheses are: i) therapy-resistant pediatric DIPG cells, including the cells that express cancer stem cell features and DIPG cell-of-origin markers, can be infected and killed by SVV-001 invitro; ii) systemic treatment of pre-established orthotopic xenograft tumors with SVV-001 would significantly prolong animal survival times in the DIPGs permissive to SVV-001; and iii) the oncolysis of DIPG tumor cells is mediated by the activation of autophagy and/or apoptosis. To test these hypotheses, we will utilize our  five newly developed orthotopic xenograft mouse models together with a recently established DIPG neurospheres by Dr. Monje et al (PNAS2011) to accomplish the following Specific Aims: 1) To determine if SVV-001 can infect and kill DIPG cells in vitro, including primary cultured DIPG xenograft tumor cells,the tumor cells that express putative brain tumor stem cell markers (CD133 and CD15) and the cell-of-origin makers (Nestin, Vimentin and Oligo 2) of DIPG; 2) To determine if intravenously administered SVV-001 can eliminate pre-formed intra-brainstem orthotopic xenografts, leading to significantly prolonged animal survival times; 3) To elucidate the mechanisms of SVV-001-induced cell killing by examining if activation of autophagy and induction of a poptosis play a role.
The innovations of our proposed studies are two folds. First, it lies in our novel use of a relatively large panel of intra-brainstem DIPG xenograft mouse models. These five models were derived from the terminal stage DIPG patients and represent the clinically proven therapy-resistant DIPGs that are in desperate need of new therapies. Since all the xenograft models were established through direct engraftment of autopsied human DIPG cells into the brainstems of SCID mice, and are shown to have replicated the histopathological and diffuse invasive phenotypes of DIPGs, they 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. We have also optimized a protocol to establish primary cultures from these DIPG xenograft tumors to facilitate the invitro screening of new therapeutic agents. Secondly, it lies in our prospective examination of the therapeutic efficacy of a novel oncolytic virus, the SVV-001, which can potentially kill the therapy-resistant DIPG cells. SVV-001 is a naturally occurring, non-pathogenic and replication-competent oncolytic virus. We have recently shown that SVV-001 is able to pass through the BBB after intravenous injection, and can effectively eliminate medulloblastoma and pediatric GBM cells, including the cancer stem cells, invivo in mouse brains. Additionally, SVV-001 kills tumor cells through infection and intra cellular replication, they may not be susceptible to the drug-and radiation-resistant mechanisms of DIPGs.
All the animal models, reagents and as says are well developed in our laboratory and we are uniquely positioned to accomplish the proposed study. Since SVV-001 is shown to be well tolerated in a recently completed Phase I trial in adult patients, and has entered phase I clinical trial in children with extra-cranial tumors,completion of our proposed study should provide strong preclinical rationale for the initiation of clinical trials of SVV-001 in pediatric DIPGs in the very near future (2-3 years).

Hypothesis- Genetically engineered mouse models of cancer are useful to elucidate mechanisms of tumorigenesis, and can serve as preclinical models for the evaluation of novel agents.  Rare tumors such as brainstem gliomas (BSGs) require genetically accurate preclinical models, which recapitulate the genetic alterations seen in the human disease, as preclinical tools. Because there are increasing numbers of available novel therapeutics, there is a need to prioritize the best combinations to translate into clinical trials. Although there have been numerous clinical trials evaluating novel agents to treat BSGs, none of them has been shown to significantly affect prognosis.  The evaluation of novel therapeutic agents as well as novel delivery routes that bypass the blood-brain-barrier (e.g. convection enhanced delivery (CED)) in such preclinical models may be predictive of responses in human clinical trials, and may result in progress against BSGs. 
 
Specific Aims  1.  To determine the in vitro activity of a PDGFR-α neutralizing antibody in cell-lines derived from PDGF-B driven BSG 
 
 2.  To evaluate the antitumor activity of systemic therapy with a PDGFR-α neutralizing antibody in the PDGF-B driven BSG mouse model 
 
3.  To evaluate the antitumor activity of convection-enhanced delivery (CED) with a PDGFR-α neutralizing antibody in the PDGF-B driven BSG mouse model   Background- BSGs account for 15-20% of pediatric brain tumors and are the leading cause of death for children with brain tumors. The median survival for these children is less than 1 year after diagnosis.  Despite decades of clinical trials evaluating novel agents to treat this disease, the natural history has not been significantly affected and 90% of children die within 2 years of diagnosis.  Involved-field fractionated radiation to a total dose of 54Gy is the current standard of care for these tumors – however, this treatment modality unfortunately provides only temporary relief of symptoms and has major side effects.    Recent genomic analysis of human BSGs have unraveled that the most commonly reported genetic alteration is platelet-derived growth factor receptor alpha or PDGFRα, which is amplified in 30-40% of BSGs and overexpressed in 67% (Becher et al. 2010, Zarghooni et al. 2010)  
 
Clinical Significance- If systemic treatment or direct treatment using convection-enhanced delivery of a monoclonal neutralizing antibody targeting murine PDGFRα demonstrates a statistically significant survival benefit in the platelet-derived growth factor-B (PDGF-B) driven BSG mouse model, we are committed to working towards translating results from this proposal into a phase I study for children with BSG.   It is worth noting that a similar neutralizing antibody from Imclone, which inhibits human PDGFRα (IC50 < 1nM), is already in clinical trials for adult gliomas as an intravenous infusion (Loizos et al.  2005), and this latter antibody can be readily translated into a phase I clinical trial to treat children with BSG. 

Patients with diffuse intrinsic pontine gliomas (DIPGs) have a uniformly dismal prognosis with a median survival of 9 months and long-term survival of less than 1%.  Radiotherapy provides only temporary improvement of symptoms.  No chemotherapy has ever proven effective. Novel therapies are desperately needed in this vulnerable population. Little was known about the biology of these tumors until recently. The availability of autopsy and some biopsy materials from children with DIPGs has finally led to a new understanding of the biology of these tumors. We are now identifying potentially important biological pathways in DIPGs that are readily targetable with currently available molecularly-targeted agents. In addition, we have successfully grown human DIPG tumors from autopsy materials in the petri dish and have developed mouse models of DIPGs – a key resource to functionally testing potential therapies. Since the number of children with this unfortunate disease is limited, and the number of available targeted agents is quite large, we hypothesize that we can identify a promising combination of molecularly-targeted agents using a functional drug screening approach.  We propose first to test the potentially effective molecularly-targeted drugs in the laboratory from DIPG tumors grown in the petri dish (Aim 1), and in mouse models of DIPG, whose biological characteristics we will first delineate (Aim 2).   We will then test the two or three most effective drugs in these models in combination. The ultimate goal is to move the most effective single agent or combination therapy forward to early phase clinical trials in the next 18-24 months. This is the first time that a group of basic and translational scientists and physicians from throughout North America have come together as a consortium to focus on DIPGs and to focus on a bench-to-bedside approach to rationally target therapy for children with DIPGs