Grants

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor wand few children with this diagnosis survive up to two years. Even with the advent of precision-based medicine, experimental therapies have yet to show benefit beyond standard radiation, highlighting the dire need to identify and investigate novel genetic therapeutic targets in DIPG. DIPG researchers now know that the patterns of mutations driving the growth of DIPG are unique, when compared to other childhood tumors and adult high-grade gliomas.  As many as 85% of DIPGs harbor mutations in histone H3 variant H3.3, coded for by the H3F3A gene – and our work and others have shown that this mutation results in critical changes in tumor biology.  It is therefore necessary to consider H3.3 mutational status when investigating novel therapeutic targets and pathways for DIPG.

Recent work by our collaborative team has demonstrated a critical genetic target in DIPG, inhibitor of DNA binding 1 (ID1). Inhibitor of DNA binding (ID) proteins are key regulators of pre-natal tissue growth and its regulation has been tied to the pathogenesis of many diseases including DIPG. Our lab has exciting preliminary data that DIPG tumor cells re-activate ID1 signaling that was used pre-natally to build the normal pons, resulting in many of the most aggressive features in DIPG.  We have also found that cannabidiol (CBD), the non-toxic and non-psychoactive member of the endocannabinoid family found in Cannabis Sativa, can reduce ID1 levels and the viability of DIPG tumor cells in vitro.  In order to define ID1 as a therapeutic target in a clinically relevant manner, it is important to establish whether ID1 can be effectively targeted in mouse models of DIPG and to understand how ID1 signaling may be regulated in different H3.3 mutational subgroups.

The experiments outlined in this protocol will answer meaningful questions about the mechanism underlying ID1-driven DIPG invasion and potentially open novel therapeutic avenues. In particular, the possible use of CBD therapy to target the ID1 pathway in DIPG is a clinically relevant but scientifically unexplored question in DIPG.

No cure has been found for diffuse intrinsic pontine glioma (DIPG), after more than 250 clinical trials.  Evidence for excessive signaling by platelet-derived growth factor receptor alpha (PDGFRA) is commonly found in biopsies of DIPG, but attempts to inhibit this protein has failed as a clinical strategy.  Based on recently published studies evaluating the RNA of individual cells, we are testing the hypothesis that only a fraction of DIPG cells are sensitive to treatments that inhibit PDGFRA signaling.  Using a technique that evaluates protein levels in single cells, this will be the first attempt to validate the previously published RNA data suggesting that multiple cell types are present within the tumor, and correlate the patterns with drug sensitivity.  With the long-term goal of deriving rational therapeutic targets from biopsy tissue, this project could improve patient selection for PDGFRA inhibition by characterizing its effects at the level of individual cells and across the different cell types within DIPG.  

Diffuse intrinsic pontine glioma (DIPG) is a malignant brainstem tumour arising in children representing a major unmet clinical need, with a 2-year survival rate close to zero. With chemotherapy ineffective and surgical intervention not possible, new therapeutic approaches are urgently required based upon the unique biological mechanisms driving DIPG tumorigenesis. We recently discovered amplification of the gene TOP3A to play an important role in a proportion of DIPGs. Critically, cells driven by this genetic alteration to be exquisitely sensitive to inhibitors of sirtuin, which appears to link TOP3A to DNA repair. We plan to explore this in more detail by turning off, and/or turning on the gene in DIPG cells, in order to better understand it’s precise function. We will also screen a range of drugs known to target sirtuins, in order to determine which may be most useful in the clinic, both in cells grown in the laboratory as well as using patient-derived mouse models. We will further look for possible combinations with sirtuin inhibitors in order to further enhance the potential for rapid translation.  

Diffuse intrinsic pontine glioma (DIPG) is a rare paediatric brain cancer that is almost universally fatal. Surgery is generally too risky and standard chemotherapy is ineffective. Radiation is the only treatment currently available with proven, yet temporary, benefits. There is no cure for DIPG. Targeted therapy uses drugs aimed at specific molecules involved in tumour growth, preventing the growth and spread of cancer cells. This approach has proven effective against several types of cancers. However, no effective targeted therapy exists against DIPG. In previous experiments, targeted therapy has been used to “switch off” a factor that is crucial for DIPG cells to grow. Surprisingly, following treatment with targeted therapy, DIPG cells not only survive but grow and thrive. A potential factor explaining this conundrum is the plasticity of brain cells. Plasticity allows brain cells to adapt to different conditions in their environment. Just like normal brain cells, DIPG cells also exhibit plasticity, and may be using it to evade drug treatments. Our plan is to use drugs that specifically inhibit DIPG’s plasticity, in combination with targeted therapies already in use for cancer treatment. This approach should enhance the anti-cancer efficacy of targeted therapies, leading to improved survival of DIPG patients. 

Our study will first be conducted in-vitro, using DIPG cells from patients, and then in-vivo, using mice as model organisms. The drugs we plan to use will target ion channels, which are pore-forming proteins found in the cell’s membrane, which control the flow of small charged molecules (like potassium) in and out of the cell. These flows of ions can rapidly change the internal wiring of a cell. For this reason, ion channels have been associated with brain plasticity, and DIPG cells are known to make extra amounts of ion channels. We will employ state-of-the-art techniques to study ion channels in a group of cancer cells obtained from DIPG patients. The techniques for studying ion channels are well established but have not been used by brain cancer researchers. Our first goal is to identify which sets of ion channels are present on the surface of DIPG cells.

Once we have identified the ion channels present in DIPG cells, we will test drugs specifically designed to block these ion channels. Importantly, drugs that block ion channels are already approved for use in the treatment of other neurological conditions such as epilepsy. Hence, these drugs have a better chance to be rapidly trialled in patients. Overall, our work aims to find new and effective treatment options DIPG patients who currently have a very poor life expectancy.

Diffuse intrinsic pontine glioma (DIPG) is the most aggressive of all childhood cancers. Standard treatment with radiotherapy is only palliative and chemotherapy has been found ineffective. Polyamines are organic compounds essential for key functions of living cells. They can be made by human cells but also generated from intestinal microorganisms and taken from dietary sources. Polyamines are produced at higher levels in cancer cells and contribute thus to aberrant growth of multiple adult and childhood tumours. Our preliminary experiments have shown that polyamines increase the growth of DIPG tumour cells. We have tested a specific combination of drugs that inhibit the production and cellular uptake of polyamines and found this to be highly active against DIPG cells but not in healthy cells. Furthermore, we have tested this combination in two separate orthotopic animal models of DIPG, which recapitulate the human disease, and found that the survival of these animals was significantly enhanced and tumour growth was reduced. This combination was also found to significantly reduce the growth of DIPG cells together with panobinostat, a targeted drug that is currently in a clinical trial for DIPG patients. This project will determine in animal models of DIPG whether polyamine inhibition therapy can enhance the effects of radiotherapy, which is the current standard treatment for DIPG patients. Furthermore, we intend to evaluate the combination of polyamine inhibitors together with panonbinostat, in animal models of DIPG.  Finally we also aim to determine the therapeutic efficacy of polyamine inhibition in other paediatric brain tumor animal models. Positive results from this study will provide the preclinical evidence required to urgently translate these novel discoveries to clinical trial to directly benefit children with DIPG and other aggressive brain tumours.

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 Intrinsic Pontine Glioma (DIPG) is a highly aggressive and infiltrating brain tumor affecting children for which there is no effective treatment.

Sequencing data have revealed  the existence of well defined subgroups of tumors based on age of onset, specific location in the brain, mutations and clinical outcome. Those tumous are characterized by specific recurrent mutations in histone genes which encodes for H3.3 and H3.1 and another mutation in a bone-related gene called ACVR1 which is largely found mutated with the histone H3.1.

Moreover, the existence of heterogeneity among the DIPG cells composing the tumor mass is increasingly evident. Our recent data suggest the presence of a “network” of distinct subpopulations that taken individually differ in their invasion and migration properties, that interact with each other and with the surrounding microenvironment to promote tumorigenesis and the progression of the tumor.

In this context, a crucial role in the communication between DIPG cells and the microenvironment may be represented by exosomes, cellular microvescicles containing RNA, DNA and proteins.

We hypothesise that the exosomes derived from DIPG cells could mediate the intercellular communication and that they could have a specific "oncogenic signature" that is specific to different molecular subgroup, and therefore could be used as an important diagnostic and prognostic marker.

To define the exosome signature of DIPG, we looked at the proteins enclosed in the exosomes and through which cells could exchange signals.

In particular, we used a panel of patient primary-primary cell lines characterized by the DIPG driver mutations (H3.3 K27M, H3.3 K27M/ACVR1 and H3.1 K27M/ACVR1) cultured in stem cell-like conditions, such as neurospheres (NS) and/or adherent to laminin (L).

Moreover, to better derive specific DIPG oncogenic exosomal signature, we used as a comparison pediatric glioblastoma (pGBM), which arise in different locations but maybe mutated in the same histone genes as DIPG.

The analysis of exosomal protein content showed that DIPG and pGBM carry unique proteins in the exosomes, highlighting the differences between the two tumor types, differences that were clearly shown also for the two DIPG subgroups H3.3 and H3.1

Focus of our study was also another molecule transported in the exosome: the micro-RNAs (miRs). Those are small RNA fragments, that may be exchanged among tumor cells and that we also considered for the identification of exosomal oncogenic signature of DIPG.

In this context, we analyzed the miRNA isolated from different patient primary-derived cell lines including different mutation and location subgroups.  Our data showed that H3.1 K27M/ACVR1 DIPG subgroup was strongly different from H3.3 K27M DIPG and pGBM, concluding that the two main histone H3 gene variants leads to distinct oncogenic programs which could be exploited to develop distinct potential therapeutic strategies.

Moreover, since it has been demonstrated that DIPG and pGBM are heterogeneous tumors characterized by the existence of different cell types that co-operate and communicate one to each other, we also isolated single cells from DIPG and pGBM primary cell lines which were later exploited to give raise to different subpopulations and studied for the exosome.

The single cell derived subpopulations were characterized for their morphology, migration and invasion properties and they were different one from each other underlying the heterogeneous nature of DIPG and pGBM tumor masses. Moreover, we also analyzed the exosome communication between tumor cells, confirming that exomes are an important key of signals exchange.

Having consolidated the role of exosomes in DIPG and pGBM cellular connections and having highlighted some important proteins as well as miRNAs, that could be considered as “oncogenic signature” of DIPG and pGBM, our further aim is to investigate their presence in the exosomes from the peripheral blood of DIPG cancer patients.

This will lead to the identification of new diagnostic markers and prognostic strategies for DIPG and pGBM tumors.

DIPG is a rare type of childhood brain cancer that is currently incurable. One barrier for progress against DIPG is the development of predictive models, i.e. models in which observations regarding the evaluation of new therapies will accurately predict observations in clinical trials for children with DIPG.   Models are important for a rare disease like DIPG as it would take a very long time to test every new potential therapy in a clinical trial for children with DIPG. While models will always be imperfect, developing improved models can accelerate the development of effective treatments for children with DIPG.  Studies using human DIPG tissue as well as postmortem human pons tissue strongly suggest that a protein called Olig2 is expressed in the original cell that acquires genetic alterations and gives rise to DIPG. Therefore, developing a mouse model that is initiated in Olig2-expressing neonatal brainstem progenitors and harbors genetic alterations commonly seen in DIPG may yield a more predictive model.  Here we propose to characterize an improved DIPG model that arises in Olig2- progenitors and to evaluate whether H3.3K27M, a genetic event present in the majority of DIPGs, influences DIPG tumor cells’ response to radiation, a treatment that children with DIPG receive.   

Children with diffuse intrinsic pontine gliomas (DIPGs) have a 2-year survival rate of less than 10%. Compared to other brain cancers, DIPG is particularly difficult to treat because it spreads behind an intact blood-brain barrier (BBB) affecting healthy developing paediatric brain tissue. There are currently no effective treatments for this disease. Although numerous novel mechanisms of biologically targeting these tumour cells are being identified in proof-of-concept studies in the laboratory, validation in living creatures and clinical application is hampered by the properties of the compounds, which are not optimised for penetration through the blood-brain barrier and into the central nervous system. We have invented an ultrasound technology – rapid short pulse (RaSP) sequencing – that delivers drugs to the developing brain with a low risk of side effects.  This will be the first demonstration of ultrasound delivery of chemotherapeutic agents to DIPG using this new technique, one that has been shown to have fewer side effects than existing ultrasound methods. The RaSP drug delivery method for DIPG has the potential to provide the urgently required improvements in the treatment of this disease in the developing brain.

The incidence of DIPG peaks at approximately 6-7 years of age, at a time of extensive myelination in the brainstem.  The ability to improve delivery of novel agents to DIPG cells, not only locally, but into cells spread through the brain, will open up a new wave of clinical trials in the disease using existing drugs without the need for further lengthy medicinal chemistry refinement.

Therapeutic ultrasound is currently engendering considerable interest because it has been shown in extensive studies that, when coupled with microbubble contrast agents commonly used for diagnostic ultrasound purposes, it can increase the spread of drugs out of blood vessels and into tumours.  This means that drugs may be infused into the blood stream, but uptake is only increased locally in the region exposed to ultrasound.  Commonly, the ultrasound beams used are focused, this allowing very selective exposure of tissue regions.  Of particular interest is the finding that ultrasound exposures can lead to a temporary opening of the blood-brain barrier (BBB), thus allowing therapeutic molecules to cross from vessels into the brain.  This is being investigated for a number of applications, including adult glioma treatments, and the first clinical trial involving Alzheimer’s drugs has just opened. The ultrasound exposure technique that we are proposing here provides the safest and most controlled changes in BBB permeability reported to date.  Compared to existing ultrasound methodology, RaSP reduces the time of blood brain barrier opening, thus reducing the risk of neuro-inflammation from blood-borne neurotoxic species such as albumin, and provides improved uniformity of drug distribution throughout the affected regions of the brain.

If this pilot study is successful we will apply for further funding to develop this technique for rapid clinical translation. Success in such a trial will further stimulate considerable interest in focused ultrasound as a technique for the treatment of brain cancers, amongst both clinicians and the general public.

In patient-derived models, targeted inhibition of ACVR1has shown some modest efficacy, suggesting that it may represent a good target for novel drug development. Despite this, little is known about the precise role of mutant ACVR1in DIPG development and/or maintenance, and there is a dearth of available immunocompetent mouse models for biological and preclinical study. Scientific Merit We have used Applied Stem Cell’s TARGATT technology to produce founders with site-specific integration of mutant ACVR1and HIST1H3Btransgenes. We plan to combine these established TET-ON HIST1H3B / ACVR1mice with novel CRISPR-basedin uterotumour suppressor gene knockout (e.g. Pten, Bcor) to generate such mouse models of this subtype of the disease. Models will be fully molecularly and phenotypically characterised, with regulable transgenes allowing for assessing the development contexts in which ACVR1and HIST1H3Bmutations interact. Bioluminescent markers are included for assessment of tumour burden in preclinical drug screening experiments. As well as providing novel insights into the role of mutant ACVR1in 6/6/2018 A MOUSE MODEL OF HIST1H3B / ACVR1 MUTANT DIPG DIPG tumorigenesis, generation of an immunocompetent model of this subgroup of DIPG will be used for preclinical screening of our ongoing candidate single agent and combination approaches. Feasibility All techniques for breeding and maintenance of genetically engineered mouse models are well established within the ICR’s Biological Services Unit and the Centre for Cancer Imaging. Within the Jones lab we have a postdoctoral research fellow with experience and expertise with the in uteroelectroporation protocols from a previous placement at University College London. All CRISPR/Cas9-based techniques are used routinely by numerous members of the lab. With the HIST1H3B / ACVR1transgenic mice already in place, combining with the these gene editing approaches is entirely feasible within the timelines of this grant.

Expertise The Jones lab is an international leader in the genomic characterisation of pGBM / DIPG samples, and has published extensively on the molecular profiling of these tumours as well as detailed functional assessment of their defining mutations. We co-discovered the presence of ACVR1 mutations in DIPG and have recently provided the first preclinical assessment of inhibitors directed against the receptor. We form part of the INSTINCT network with Great Ormond Street Hospital and Newcastle University, and the CRUK Children’s Brain Tumour Centre of Excellence (with the University of Cambridge), particularly focussed on drug development for high risk paediatric brain tumours. Chris Jones is biology lead on the HERBY and BIOMEDE clinical trials, and former Chair of the Biology Subcommittee of the SIOPE HGG / DIPG Working Group, allowing rapid dissemination of results and clinical translation.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are devastating pediatric brainstem tumors that lack effective treatment and are uniformly fatal. Patient studies have identified recurrent genetic lesions that drive the development of these tumors. Almost all DIPGs carry mutations in genes encoding either replication-dependent histone-H3 proteins (mostly HIST1H3B) or in a replication-independent histone (H3F3A). These mutations always substitute lysine with methionine at position 27 (K27M) of the H3 protein. The tumorigenicity of histone K27M mutations is thought to stem from epigenetic reprogramming of tumor-initiating glial cells in the brain. Moreover, recent genetic data strongly suggest that DIPGs can be clustered into distinct subtypes based on specific “partner” mutations that co-occur with the K27M H3 mutations. For example, most H3F3AK27M mutant tumors carry lesions in the well-characterized tumor suppressor gene TP53. This is not the case for HIST1H3BK27M mutant malignancies, which instead commonly harbor lesions that either cause a gain-offunction in ACVR1, a bone morphogenetic protein (BMP) type I receptor, or hyperactivate the PTEN/PI3K pathway. The oncogenic mechanisms of action of activating mutations in ACVR1 are poorly characterized. Understanding the mechanisms that drive DIPG subtype development, and how these tumors might differ in therapeutic vulnerability, is crucial for the development of effective DIPG treatments.  
 
In our proposal, we will test the hypothesis that oncogenic synergy between epigenomic reprogramming induced by HIST1H3BK27M mutations and cellular hyperproliferation driven by ACVR1 and PTEN/PI3K pathway mutations underlie unique therapeutic vulnerabilities in DIPG tumors. We will deploy a multidisciplinary approach that combines complementary areas of expertise and reagents, including the generation and analysis of the first pre-clinical mouse models harboring DIPG-causing mutations in the endogenous Acvr1 and Hist1h3b genes. Using these models, we will characterize the molecular effects of Acvr1 and Hist1h3b mutations and dissect their interaction and synergy. We will then harness an innovative direct in vivo CRISPR/Cas9 platform to combine multiple co-occurring mutations and describe their oncogenic mechanisms of action. We will pay particular attention to investigating how PTEN/PI3K pathway hyperactivation cooperates with Acvr1 and Hist1h3b mutations. Finally, we will meld these analyses with human DIPG transcriptome data and perform functional experiments in patient-derived cell lines to uncover candidate therapeutic targets. We have already established a broad toolbox of reagents useful for our planned studies and have accumulated substantial preliminary data in support of our objectives.  
 
We expect that our project will uncover the molecular mechanisms whereby ACVR1, HIST1H3B and PTEN/PI3K pathway mutations cooperate to drive DIPG development and progression. We further anticipate that our studies will reveal candidate therapeutic targets for tumors harboring this combination of lesions, and possibly for DIPGs in general.  

We and others recently discovered a novel cancer gene, ACVR1, to be mutated in approximately 25% diffuse intrinsic pontine glioma (DIPG), most commonly in the youngest patients, and co-segregating with K27M mutations in histone H3.1 (HIST1H3B/HIST1H3C). ACVR1 encodes a receptor serine/threonine kinase mutated in the germline of patients with the congenital malformation syndrome FOP (fibrodysplasia ossificans progressiva), and is known to activate the BMP pathway via aberrant responsiveness to activin A and other ligands. As a proof-of-principle to explore the efficacy of targeting the receptor in DIPG, we have utilised a novel series of inhibitors developed by the Structural Genomics Consortium for FOP in our patient-derived models. We have shown a differential efficacy in ACVR1 mutant DIPGs both in vitro and in vivo in response to the compounds LDN-193189 and LDN214117, representing distinct chemotypes, and with concurrent downstream pathway inhibition. Although both compounds penetrate the CNS at doses able to elicit a response, the effects on survival in our orthotopic xenografts remains modest, with an extension of only 14 days. We hypothesis that combining ACVR1 inhibitors with other agents will lead to a prolonged response that may be significantly more likely to prove beneficial to children in the clinic. We propose to identify and test the most effective combinations through rational candidate and screening approaches in vitro and in vivo. This will be underpinned by our expertise in disease biology and genomics, as well as an innovative analytical approaches to identify novel interactions.

We propose a phase I/limited efficacy clinical trial investigating repeated administration of MTX110, a soluble form of panobinostat, given via convection-enhanced delivery (CED) to pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG). We hypothesize this therapy will be safe and well-tolerated and will be efficacious and prolong survival compared to historical controls.

The survival outcomes for pediatric DIPG are dismal. Despite decades of clinical trials and multi-modal therapy, there are essentially no survivors of this devastating disease. The location of DIPG within the central nervous system (CNS) and, more specifically, within the brainstem present unique treatment challenges. The presence of the blood brain barrier limits systemic delivery of therapy from reaching therapeutic levels in the tumor. Further, location within the brainstem prevents surgical resection. Because of this, delivery of drug to DIPG tumors via novel strategies is both warranted and likely necessary to improve outcomes. MTX110 given via CED offers such a strategy. CED uses a catheter system implanted within the tumor that delivers drug directly to the tumor along a pressure gradient. This direct tumor delivery strategy offers drug distribution throughout the tumor and avoids the toxic side effects often seen with oral or intravenous systemic drug delivery.

Panobinostat is a pan-histone deacetylase inhibitor currently approved for treatment of multiple myeloma and having shown pre-clinical efficacy in DIPG cell lines and animal models, regardless of histone status. MTX110 is a novel, soluble formulation of panobinostat that can be given intratumorally to DIPG tumors via CED. Our collaborators have shown in small as well as large animal studies (pig) that CED of MTX110 is feasible and safe. At UCSF, we have treated one patient under compassionate use with 2 CED treatments of MTX110 with no safety concerns. In the UK, several subjects have been treated with MTX110 via CED; one subject has continued on this regimen for several months.

In this study, we aim to investigate the safety and early efficacy of repeated administrations of MTX110 given via CED to children with newly diagnosed DIPG that have completed standard-of-care focal radiotherapy. To complete this investigation, we will carry out a phase I/limited efficacy clinical trial at a minimum of two institutions: University of California, San Francisco (UCSF) and Memorial Sloan Kettering (MSK). The trial will follow an accelerated titration design (ATD) that allows for intra-patient dose escalation and potentially decreases the number of patients treated at sub-efficacious dose levels. The trial includes 5 doses levels, each with increasing volume of drug and therefore, total drug dose. The ATD allows for transition to a standard 3+3 design within each dose level, should toxicity occur. Once the phase I dose escalation is complete and the recommended phase II dose determined, the trial will move into an expansion cohort to assess efficacy based on overall survival at 12 month (OS12). The primary aim of our investigation is to determine safety and toxicity of repeated administration of MTX110 delivered via CED to our target population. This aim will be assessed by monitoring adverse events, laboratory assessments, and physical examinations for each subject that receives at least 1 dose of drug. Descriptive statistics will be used to summarize the toxicity data. The secondary aim will investigate the efficacy of this approach by assessing OS12 and compare to historical controls using KaplanMeier survival analyses. The null hypothesis is OS12 of 40%, the alternative hypothesis is OS12 of 60%. An exploratory aim will also use quality of life assessments to evaluate the impact of this treatment approach on the quality of life for our patient population. Descriptive statistics will be used to summarize all quality of life data.

By completing the trial at multiple institutions, our study will increase patient catchment, leverage significant expertise with this novel delivery strategy and avoid barriers in meeting the anticipated accrual goal of 24 patients. The clinical trial will be executed by the Pacific Pediatric Neuro-Oncology Consortium (PNOC). PNOC has extensive experience executing multi-institutional clinical trials and offers the appropriate infrastructure to conduct multi-site studies including a secure HIPAA protected database and central monitoring by the UCSF Cancer Center’s data and safety committee. The industry partner, Midatech Pharma, is committed to completing this trial alongside our group and will provide study drug for free.

Sabine Mueller, The Regents of the University of California, $100,000

Diffuse    Intrinsic    Pontine    Glioma    (DIPG)    is    a    devastating    tumour    that    occurs    predominantly    in    young    children    and    results    in    a    near    100%    fatality    rate    within    2    years    of    diagnosis.    Its    diffuse    growth    pattern    and    eloquent    location    precludes    surgical    resection.    Numerous    clinical    trials    of    chemotherapeutic    agents    have    failed    to    demonstrate    an    improvement    in    prognosis    or    survival.    Our    current    best    standard    of    care    is    radiation    therapy    which    provides    temporary    relief    of    symptoms    and    minimal    gains    in    life    expectancy.        
In    recent    years,    greater    understanding    of    the    molecular    landscape    of    DIPG    has    resulted    in    the    development    of    exciting    new    molecular    therapies    and    sophisticated    pre-clinical    models.    Drug    delivery    however,    remains    a    major    challenge    due    to    the    blood    brain    barrier    (BBB).    To    circumvent    this    obstacle,    we    propose    the    use    of    Magnetic    Resonance    Image-guided    Focused    Ultrasound    (MRgFUS)    to    transiently    open    the    BBB    without    tissue    injury.    Intravenously    administered    microbubbles    prior    to    focused    ultrasound    (FUS)    treatment    results    in    a    mechanical    interaction    between    ultrasonic    waves,    injected    microbubbles    and    the    capillary    bed    resulting    in    enhanced    permeability    and    a    window    of    opportunity    for    drug    delivery.        
Through    an    ongoing    collaboration    with    Dr    Meaghan    O’Reilly    of    the    focused    ultrasound    laboratory    at    the    Sunnybrook    Research    Institute,    we    have    successfully    demonstrated    both    the    safety    of    MRgFUS    in    the    rodent    brainstem    and    the    ability    to    concentrate    an    intravenously    administered    chemotherapy    agent    (Doxorubicin)    into    the    region    (Fig.    C-H    &    J).    We    now    wish    to    conduct    the    next    steps    required    for    the    clinical    translation    of    MRgFUS    as    a    method    of    drug    delivery    in    DIPG.    As    such,    our    aims    are    as    follows:    
Aim    1:    To    identify    clinically    available    drugs    that    target    DIPG    cell    lines.    This    will    include    identifying    agents    that    show    efficacy    as    monotherapies    as    well    as    combination    agents    that    act    in    synergy.        Aim    2:    To    demonstrate    the    efficacy    of    intravenously    administered    chemotherapeutics    when    combined    with    MRgFUS    in    the    treatment    of    pre-clinical    models    of    DIPG.        
    
Working    in    conjunction    with    the    Ontario    Institute    of    Cancer    Research,    we    will    have    access    to    several    hundred    clinically    available    drugs    which    we    will    use    to    conduct    a    high    throughput    drug    screen    on    multiple    DIPG    cell    lines.    From    this    screen,    we    will    select    effective    single    agent    therapies    as    well    as    drug    combinations    that    demonstrate    synergy.    Having    identified    the    most    effective    agents,    we    will    test    these    in    a    genetically    engineered    mouse    model    (RCAS-Tva    PDGFRA-driven    DIPG    model)    as    well    as    an    orthotopic    xenotransplantation    model    (stereotactically    injecting    SU-DIPG    VI    cells    into    the    pons    of    immunocompromised    mice).    We    will    then    quantitatively    measure    (using    mass    spectrometry)    the    delivery    of    drug(s)    to    the    tumour    in    addition    to    parameters    of    in    vivo    therapeutic    efficacy.        
By    re-evaluating    exisiting    chemotherapeutics    and    demonstrating    their    efficacy    in    combination    with    MRgFUS    delivery,    we    could    open    the    gateway    of    potential    therapeutic    options    to    children    afflicted    with    the    disease.    With    this    information    and    our    ready    access    to    a    clinical    focused    ultrasound    delivery    device    we    would    be    poised    to    offer    the    first    ever    Phase    I/II    safety    and    efficacy    trial    of    an    MRgFUS    delivered    treatment    in    DIPG.    

The objective of this application is to demonstrate that combining MCB-613, a small molecule stimulator of the oncogenic steroid receptor co-activator (SRC), with ionizing radiation would synergistically kill tumor cells of diffuse intrinsic pontine glioma (DIPG) in vivo and significantly prolong survival times in patient tumor-derived orthotopic (intra-brain stem) xenograft (PDOX) mouse models. This proposal is a follow-up study from a preclinical analysis of MCB-613 on DIPGs that was generously funded by the Cure Starts Now Foundation and DIPG Collective (2015-2017). Our goal is to establish strong preclinical rational to support a rapid initiation of MCB-613 clinical trials in children with DIPG.  DIPG is the most lethal childhood cancer, and virtually all children with this disease die within 1-2 years of diagnosis. In our previous study that was generously funded by the Cure Starts Now Foundation and the DIPG Collective, we have shown that MCB-613 can overcome some major challenges for the development of new therapies for DIPG using our panel of intra-brain stem PDOX models of DIPG, including 1) strong antiproliferative activities in vitro in traditional monolayer cells as well as in neurospheres (enriched with the putative cancer stem cells), 2) low toxicity to normal cells in NOD/SCID mice that were treated for 4 weeks;  3)  capability of passing through the blood brain barrier (BBB) to reach xenograft tumor cells in vivo in mouse brain stems; 4) significant prolongation of animal survival times (the gold standard of therapeutic efficacy) in a DIPG PDOX model acting as single agent; and 5) synergistic killing of DIPG xenograft cells and significant prolongation of animal survival times when combined with fractionated radiation. There were, however, some missing data that are critically needed to move MCB-613 into clinical trials. When administered as single agent, MCB-613 was active only in 1/4 DIPG models and the synergistic cell killing was only demonstrated in one DIPG model. Fortunately, it was also during the previous funding period that we have identified the “under treatment” (i.e., short and insufficient drug exposure time of 14 days) as one of the causes of tumor progression.  Our central hypothesis for this proposal is that 1) Increase the length of  MCB-613 treatment time and in combination with clinically relevant fractionated ionizing radiation will  synergistically kill DIPG cells in vivo in intra-brain stem DIPG xenograft models and significantly prolong animal survival times compared to mice treated with MCB-613 and with radiation alone; and 2) analyzing multiple DIPG xenografts that exhibited differential responses would facilitate the discovery of new diagnostic marker(s) for patient selection.   To test our hypothesis, we will utilize our established PDOX models of DIPG to accomplish the following Specific Aims: 1) Demonstrate that the combined treatment of intra-brain stem DIPG xenografts with extended MCB-613 treatment (> 4 weeks) and fractionated radiation (2 Gy/day x 5 days) will synergistic improve therapeutic efficacy and significantly prolonged animal survival times. 2) Understand mechanisms of the synergistic cell killing induced by the combination of MCB-613 and XRT in DIPG cells. 3) Identify the cellular and molecular cause of therapy resistance toward the combined MCB-613 and XRT treatment.    This proposal is innovative on several levels.  Firstly, our panel of intra-brain stem DIPG PDOX mouse models is derived from terminal-stage DIPG patients; consequently, they represent therapy-resistant disease, which is in desperate need of new therapies.  These models provide us with unprecedented opportunities to study tumor biology and test new therapies in vivo in a microenvironment closest to human DIPGs.  Secondly, our research proposal is the evaluating the therapeutic efficacy of MCB-613, a novel compound with a novel mechanism of action.  This drug crosses the BBB and accumulates in brain tissue, thus allowing MCB-613 to research the tumor site. Indeed, MCB-613 has already shown promising anti-tumor activities against DIPG cells both in vitro and in vivo. Most importantly, since our treatment approach combines MCB-613 with radiotherapy, the standard treatment for DIPGs, our chances of rapid translation of the drug into clinical trials are greatly improved.