Grants

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.




Texas Children's Cancer Center - $100,000

Amount
$100,000.00
Date

November 2013

Targeting DIPGs in vivo in Intra-brain Stem Xenograft Mouse Models through Combined Radiation and Oncolytic Viral Therapy.

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.

Children's National Medical Center - $99,979

Amount
$99,979.00
Date

November 2013

Comparative Proteomic and RNA Sequencing of DIPGs

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.

Texas Children's Cancer Center - $100,000

Amount
$100,000.00
Date

October 2012

Preclinical Evaluation of Novel BMI-1 Inhibitors In Vivo in Intra-Brainstem Orthotopic Xenograft Mouse Models

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.

St. Jude's Children's Research Hospital - $15,000

Amount
$15,000.00
Date

October 2012

Establishment and Characterization of Renewable Tissue Resources for DIPG Research

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.  

Texas Children's Cancer Center - $100,000

Amount
$100,000.00
Date

November 2011

Eliminating Therapy-Resistant Diffuse Intrinsic Pontine Gliomas with Oncolytic Picorna Virus SVV-001

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).

Duke University - $82,049

Amount
$82,049.00
Date

November 2011

Preclinical Evaluation of Systemic and Direct Delivery of a PDGFR-Alpha Antibody

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. 

Doernbecher Children's Hospital - $100,000

Amount
$100,000.00
Date

November 2011

Rapid Preclinical Development of a Targeted Therapy Combination for DIPG.

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