Grants

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.  

Backgound. Radiotherapy is still the mainstay of the treatment for DIPG. If the majority of children experience an improvement of their neurological condition following irradiation, this effect is not observed in all patients and is universally only transient. Determinants of the response to radiotherapy have yet to be defined. We have described two distinct forms of DIPG according to the type of histone H3 mutated with different response to therapy and survival (Castel et al., Acta Neuropathologica 2015 & 2016). In particular, in a cohort of 67 patients treated at our center, clinical response to radiotherapy was shown to be better in children with DIPG harboring a K27M mutation in HISTH3B gene than in children with a K27M mutation in H3F3A gene (85% vs. 55.3%, p=0.0263, Chi Square test). These data need to be confirmed in a larger study and refined thanks to the availability of DIPG models, to be able to predict response to radiotherapy more accurately. Hypothesis. We hypothesize that radioresistance of DIPG is first an intrinsic phenomenon than can be explained by the genetic background of the disease and second an acquired phenomenon that we aim to describe as oligoclonal or polyclonal. Design and methods. The project proposal aims to bridge biological studies on relevant preclinical models and appropriate patient’s data. The project will take advantage of a unique cohort of biologically defined DIPG patients with full clinical follow-up and treated at a single center as well as a large set of both in vitro and in vivo preclinical models. We have recently published the methods used to generate them and their relevance with respect to the biology of DIPG (Plessier et al., Oncotarget 2017).  We will treat our panel of cellular and murine models with irradiation in order to:  (i) correlate their radiosensitivity with their molecular profile (type of histone H3 mutated at K27 residue and subclonal driver alterations),  (ii) correlate these findings with patient’s data,  (iii) model and depict in vitro and in vivo the molecular aspects of radioresistance and tumour escape from radiotherapy using barcoding and lineage tracing. DIPG stem cells will be infected with lentiviruses encoding fluorescent proteins (a combination of 3 different markers) or unique barcodes. The fluorescence will be used to retrieve the tumor cells by cell sorting and to trace their evolution and development in the mouse brain after clarification. The unique barcodes will be identified by NGS allowing to quantify the clonality of the radioresistance.  Intrinsic mechanisms of radioresistance will be identified by correlating response to radiotherapy and molecular background both in the preclinical models and in the patients’ cohort. Acquired mechanisms of resistance will be studied by cell tracking after lentiviruses transduction and next-generation sequencing. Clinical significance. This project will determine molecular biomarkers associated with radioresistance of DIPG that will be useful in patient’s management. New therapeutic targets will be defined based on association of genetic alterations with initial radioresistance, or acquired genetic modifications after radiotherapy. 

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor which affects children and for which there is no effective treatment at the moment.  We and others have recently shown that DIPG is characterized by a remarkable degree of intratumoral heterogeneity and consists of distinct genetically and phenotypically heterogeneous subclonal populations. It is well recognized that exosomes mediate the cross-talk among the tumor cells and between the tumor and its microenvironment. We hypothesize that the DIPG subclonal network is sustained through paracrine signaling and that exosomes are involved in this intratumor crosstalk promoting tumorigenesis and tumor progression. To understand the role that exosomes play in DIPG tumorigenesis and determine the mechanistic basis of the exosome-mediated interclonal communication, we aim to: i. determine the specific DIPG-exosome signature within the distinct mutational subgroups and within distinct sub-clonal populations; ii. define the exosome-mediated mechanisms of crosstalk between distinct DIPG subclones and determine the functional consequences of such uptake. To address these aims we will specifically isolate and characterize exosomes derived from different DIPG patient primary-derived cells using proteomic and miRNA analysis. These exosomal “signature” will be compared with exosomal signature of glioblastoma patient’s primary cells. Furthermore we will investigate in details the specific profile of exosomes derived from DIPG subclonal populations and we will analyze the functional consequences (effect on growth and invasion) of exosomal uptake in co-culture experiments of DIPG subclones. Our goal is to better elucidate the mechanisms of cell-cell communication that mediate the DIPG growth and invasion.  We believe that this study will lead to the identification of potential new targets and the development of new diagnostic/prognostic tools for patients affected by DIPG. 

Malignant brain tumors are the leading cause of cancer-related mortality in children (1), and diffuse intrinsic pontine glioma (DIPG) is one of the most devastating, with a median survival of <1 year following treatment with radiation therapy (2). Despite more than 250 clinical trials over the past 30 years (3), not a single chemotherapeutic agent has demonstrated survival benefit. There is thus an urgent need for novel treatments for this disease. Our data and two recent high profile studies have implicated the protein EZH2 (enhancer of zeste homologue 2) as a specific therapeutic target in DIPG (4,5). Although EZH2 inhibitors exist and have shown very promising results in phase I clinical trials in other cancers, all the currently available EZH2 inhibitors do not cross the blood brain barrier and thus cannot be effectively employed in brain tumor patients.  
 
We have generated novel brain penetrant inhibitors of EZH2 in conjunction with the drug development institute at MSKCC (Tri-Institutional Therapeutics Development Institute), and the major goal of this project is to validate the in vivo efficacy of these inhibitors using pre-clinical models of DIPG. 
 
We hypothesize that brain penetrant inhibitors of EZH2 will represent effective therapeutics for DIPG based on our data, and recent studies (4,5) which show genetic knockdown of EZH2 has an anti-tumor effect in DIPG. 
 
The primary objectives of this project are: 1) Validate the in vivo efficacy of novel brain penetrant EZH2 inhibitors we have generated, using preclinical models of DIPG  2) Characterize critical epigenetic effects of histone mutations (found in DIPG) on self-renewal and differentiation, to gain understanding of how these mutations promote tumors 
    
Impact and Innovation: Aim 1 is directly translational, and our aim is to have a small molecule to enter IND (investigational new drug) studies in preparation for phase 1 clinical trials in patients with DIPG. EZH2 has been strongly implicated as a target in other brain tumors e.g. CNS atypical teratoid rhabdoid tumors, and various types of brain metastases; thus successful development of a brain penetrant EZH2 inhibitor will also be beneficial for this group of patients.  Aim 2 is directly mechanistic; and we anticipate that the proposed experiments will reveal insights into how H3K27M mutations promotes the formation of tumors.  
 
Feasibility: All the techniques outlined in this proposal, are already well established in the Allis Laboratory and will allow prompt initiation and completion of the project. It is anticipated that further optimization of the novel brain penetrant EZH2 inhibitors will be required, and further chemical biology support for continued optimization of the already generated brain penetrant inhibitors will be provided by the Tri-Institutional Therapeutics Discovery Institute (Tri-I-TDI). The ultimate goal will be to have a small molecule to enter IND (investigational new drug) studies within a period of 12-18 months in preparation for phase 1 clinical clinical trials in DIPG patients. 
 
Expertise: This project is a collaboration between Dr. Richard Phillips is a brain tumor specialist and physicianscientist at MSKCC and Dr. David Allis a world-leader in the study of epigenetics at Rockefeller. The Allis Lab has been a leader in dissecting the mechanisms by which the H3K27M mutation promote DIPG (6,7). Dr. Richard Phillips MD PhD has spear-headed a collaboration with chemical biology experts at the MSKCC drug development core (Tri-I-TDI) which has led to the development of two novel brain penetrant inhibitors of EZH2 which will be further characterized in this study.   The combination of these expertise will facilitate the successful completion of this project. 

Single-cell genomic profiling is revolutionizing our understanding of tumor biology, as it enables for the first time a genome-wide interrogation of tumor programs at cellular resolution, offering unprecedented insights into tumor cells and their micro-environment at a depth that was unthinkable even a few years ago. I expect that defining the genetic and cellular programs of DIPG with these techniques directly deployed in patient samples will reveal the key programs that underlie DIPG biology. By additionally leveraging the CRISPR/cas9 system to perform targeted and specific genetic knock-outs, we will functionally test candidate regulators and expect to identify novel tumor vulnerabilities and dependencies.  
 
Disease Impact   Applying single-cell genomic technologies to precious patient-derived tumor samples at biopsy as well as autopsy will shed unprecedented light on unique tumor vulnerabilities and dependencies that are not identifiable by bulk genetic studies alone. The proposed research will provide an unparalleled view of the cellular architecture and transcriptional networks underlying DIPG biology, and moreover, reveal novel tumor vulnerabilities that could rapidly enter pre-clinical and clinical trials.  
 
Innovation   The proposed study is the first ever to systematically study DIPG at cellular resolution, and is expected to reveal the interplay between genetically and developmentally driven programs in DIPG. By using the combination of cutting-edge single cell genomic technologies, genome editing tools and computational analysis available at our institutions and at the Broad Institute, these novel and complementary approaches will shed light on the molecular pathways driving DIPGs.  
 
Feasibility  Single-cell RNA sequencing as well as genome editing technologies have been established in the laboratory of Dr. Suva.  A close collaboration with computational scientists at the Broad Institute is also already in place.  The novel findings contributed by this study will provide a rational basis for renewed attempts at improving clinical care of DIPG patients. 
 
Expertise  Dr. Suva and Dr. Filbin have unique and complementary expertise that makes them the ideal investigators to complete the proposed research.  Dr. Filbin is a pediatric neuro-oncologist at Dana-Farber Cancer Institute and research fellow in Dr. Suva’s laboratory at Massachusetts General Hospital (MGH) and the Broad Institute.  Her previous work includes the discovery of a novel combinatorial targeted treatment for glioblastoma, which led directly to clinical trials in adults and pediatric patients with gliomas.  Dr. Suva is a faculty scientist and neuropathologist at MGH and the Broad Institute and has led ground-breaking research deploying single-cell genomics in adult gliomas. 

Diffuse intrinsic pontine gliomas (DIPG) are infiltrative, highly aggressive pediatric brainstem tumors with limited therapeutic options. Despite international efforts to improve outcome, DIPG show poor response to conventional radiation and chemotherapeutic strategies. Only within the last decade have studies really begun to decipher the molecular mechanisms behind DIPG tumorigenesis, with the goal of identifying novel therapeutic targets for this lethal disease. Next generation sequencing studies of DIPGs have identified canonical mutations, most frequently K27M substitutions in either H3F3A (H3.3) or HIST1H3B (H3.1) as well as TP53, ACVR1, PIK3CA and PDGFRA, among others. These results highlight the importance of epigenetic dysregulation in the pathogenesis of DIPG. However, the fact that H3K27M tumors typically harbor additional genetic aberrations and that modeling efforts using histone mutations alone have failed to induce transfomation, strongly supports the notion that H3K27M per se is likely insufficient to drive malignant transformation.  Additional mechanisms likely exist and are essential for DIPG commencement and/or progression beyond epigenetic and genetic modifications in nDNA.       Unlike normal cells, cancer cells have the capability to make an energy adaption through metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and other metabolic pathways. This occurs regardless of oxygen abundance (the Warburg effect) in order to facilitate their proliferation particularly under unfavorable microenvironments. Abnormal OXPHOS and aerobic metabolism as a result of mitochondrial dysfunction have long been hypothesized to contribute in diverse ways to the multistep process of tumor progression. In recent years, a large number of somatic mutations in the mitochondrial genome (mtDNA) and aberrant mtDNA amount have been increasingly detected in a broad spectrum of primary human cancers. Due to decreased expression of mtDNA-encoded polypeptides and impaired function of respiratory enzyme complexes, quantitative change in mtDNA may decrease mitochondrial respiratory activity and lead to persistent defects in the OXPHOS system. Decreased mtDNA copies and defective mitochondrial function have been strongly linked to neoplastic transformation, tumor progression, metastasis, chemo/radioresistance, and poor prognosis in several types of solid tumors. Our recent work funded by the DIPG Collaborative demonstrated that somatic mtDNA mutations and reduced mtDNA content are highly frequent events in DIPG.  Moreover, partial depletion of mtDNA to DIPG-like levels in immortalized NHA (iNHA) cells significantly increased tumorigenicity in vivo. These findings led us to hypothesize that mitochondrial dysfunction and incompetent oxidative metabolism owing to lower mtDNA copies by themselves, or in a coordinate fashion with nDNA alterations, may be involved in DIPG tumorigenesis and DIPG cells may be characterized by “Warburg rearrangement for metabolism”. Work proposed in this study aims to revert the Warburg metabolism and rebuild normal OXPHOS activity in a panel of patient-derived primary DIPG cell lines by targeting reduced mitochondrial number through pharmacological stimulation of PGC-1α-mediated mitochondrial biogenesis (Aim 1). The potential in vivo efficacy of three mitochondrial biogenesis drugs (AICAR, resveratrol and metformin) that have excellent profiles and are being used or trialled to treat mitochondrial disorders in other human diseases (e.g. Alzheimer's disease, Type 2 diabetes) will be evaluated in both transgenic murine and human DIPG xenograft models. Through the use of high-throughput synergy drug screening, we will produce preclinical data identifying the best clinically approved compounds to be used in combination with mitochondrial targeting in the chemoadjuvant setting (Aim 2). Our innovative approach of manipulating mtDNA quantity to correct compromised mitochondrial function thereby reversing the malignant phenotype of DIPG, either alone or in combination with conventional therapies, will yield important preclinical data for designing novel therapeutic strategies. Our group is a leader in the field of DIPG and being part of the largest pediatric neuro-oncology team in Canada and active contributors in the DIPG collaborative, we are ideally situated to translate discoveries made through this project into phase I/II clinical trials for this devastating pediatric cancer.   

DIPG is characteristically infiltrative (i.e. diffuse and intrinsic), and this infiltrative/invasive behavior is destructive both in the brainstem and in other areas of the central nervous system to which DIPG spreads during the course of the disease. In work previously funded by the Cure Starts Now and the DIPG Collaborative, we discovered that neuronal activity promotes DIPG cell invasion through activity-regulated secreted factors that includes an endogenous antagonist to the Nogo receptor (NgR). NgR signaling is a key mechanism that restricts the plasticity and regeneration of normal brain cells, and blocking the Nogo pathway results in increased motility of cells and cellular processes such as axonal outgrowth. In the normal brain, this signaling pathway plays a role during early brain development and may continue to play a role in ongoing brain plasticity. DIPG cells express NgR and in preliminary studies we have found that blocking NgR signaling with recombinant antagonist or by deleting the NgR gene via CRISPR gene editing from patient-derived DIPG cells dramatically increases DIPG invasion in vitro. In the proposed studies, we will expand these observations to a larger number of patient-derived DIPG cell cultures to determine how universal this mechanism may be, and will assess the importance of NgR signaling to DIPG invasion in vivo using genetic models. If NgR signaling proves to be an important mechanism controlling DIPG invasion, then stimulating the NgR receptor may be an innovative therapeutic strategy to control infiltration of DIPG cells throughout the brainstem and prevent spread more diffusely to the cerebrum and spinal cord.

The regulation of the NgR signaling by neuronal activity represents one of the many ways that experience shapes brain development and plasticity. The effects on DIPG invasion represents yet another way that DIPG cells hijack normal mechanisms of brain development to promote disease progression. Ultimately, by understanding the ways DIPG subverts mechanisms of childhood brain development and adaptability, we hope to develop effective and tumor-specific strategies to disrupt the ability of the tumor cells to use these crucial signals in the tumor microenvironment.

Whole genome sequencing on DIPG biopsy and autopsy samples have identified specific mutations in Histone H3, ACVRI, TP53 and ATRX causing heterochromatin silencing, genetic instability and alterations in DNA damage response pathways. Using a unique panel of patient derived DIPG cultures, we have further examined the molecular profile of DIPG to understand key oncogenic drivers within these tumours and identify novel therapeutic targets. Excitingly, we have found overexpression of multiple regulators which control the cell cycle. Of these, Polo-like Kinase 1 (PLK1) represents the most promising cell cycle factor for targeting in DIPG. PLK1 not only activates the key intermediaries which promote tumour cell growth and division, but also controls cellular response to DNA damage, inducing cell cycle arrest to promote DNA repair and avoid cell death while regulating the mechanisms used to escape arrest and continue tumour growth. Importantly, we hypothesise the rapid repair of DNA and recovery from cell cycle arrest controlled by PLK1 may account for the resistance to radiotherapy commonly seen in children with DIPG. We propose that inhibiting PLKI activity may impede cell cycle progression, block DNA repair and recovery following cell cycle arrest, radiosensitise the tumour and increase the levels of single stranded DNA above the threshold required to induce DNA lethality resulting in tumour cell death. We also hypothesise that the activity of PLK1 inhibitors may be synergistically enhanced through the development of rational combination therapies.

Our data shows PLKl is essential for DIPG tumour cell growth and that PLKl inhibition provides one of the most promising potential treatment applications for children with DIPG to date. We have found single agent PLK1 inhibition using clinically available therapies has extremely potent cytotoxic effects against DIPG cultures in vitro, with near complete eradication of DIPG cells, inhibition of colony formation and a significant induction of G2 cell cycle arrest all at exceptionally low nanomolar potency (IC50 <10nM). Using our most aggressive patient-derived in vivo model of DIPG, we have examined the activity of 2 different PLKl inhibitors as single agents and found that both therapies significantly and potently enhanced the survival of all treated animals (P=<0.0005). Importantly, PLKl inhibitors have shown the most potent anti-DIPG effect in vitro and in vivo compared with any other therapy we have tested in our models to date. We have also found that PLKI inhibition targets one of the major oncogenic drivers in DIPG, the Phosphoinositide 3-kinase (Pl3K) pathway. We have preliminary evidence PLK 1 inhibition is synergistically enhanced upon addition of the Pl3K inhibitor BKM120, providing a highly potent combination therapy for DIPG (Combination Index 0.0004). We have seen a similar effect of PLK1 inhibitors in combination with Temsirolimus (an inhibitor of Mechanistic Target of Rapamycin). As a result, we seek to conduct an analysis of PLK1 inhibitors in combination with inhibitors which target key oncogenic pathways in DIPG to identify the two most potent and synergistic combinations, their mechanism of action and their efficacy in our two orthotopic models of DIPG, providing the necessary quantum of data to move these treatments to the clinic.

Our team has the necessary skills and expertise to ensure success of the proposed project and successful clinical translation. PI Franshaw is an early career Research Officer at the Children's Cancer Institute with expertise in establishing and maintaining DIPG cultures, drug discovery, in vivo models of DIPG, advanced imaging and cancer cell biology. Since joining the Institute late 2012, she has helped establish multiple DIPG cultures, 2 in vivo models of DIPG and identified multiple novel treatments against DIPG currently under investigation. Her most recent discovery of the potency of PLKI inhibitors against DIPG is one of the most promising findings in the field to date. Co-PI Ziegler is a paediatric oncologist and director of the clinical trials unit at the Kids Cancer Centre (Sydney Children's Hospital), Group Leader at the Children's Cancer Institute and a Conjoint Associate Professor at the University of New South Wales. He is a national leader in early phase clinical trials in paediatric oncology, testing novel therapeutic agents in children with relapsed and refractory malignancies for whom there are no known effective treatment strategies. Support by A/Prof Ziegler will ensure success of the project and the rapid movement of effective therapies to the clinic to directly benefit children with DIPG.

Validation of TAK228 as an Effective Drug for Treating Children Diagnosed with DIPG Diffuse intrinsic pontine glioma (DIPG) is an untreatable childhood cancer.  DIPGs develop in pons making them surgically unresectable.  We and others have identified mutations in histone and their obligate partner mutations that seem to drive tumorigenicity. While epigenetic drugs targeting histone mutation have shown positive outcome in preclinical setting, these drugs have yet to show effectiveness in the clinic.  Moreover, >70% of DIPGs are driven by AKT gain or PTEN loss.  Our collaborative team at Children’s National and Johns Hopkins School of Medicine have achieved robust preliminary success in targeting AKT and PTEN pathways using TAK228, also known as sapanisertib, MLN0128 and INK128. We also find that TAK228 in combination with radiation leads to increased DIPG apoptosis. We also find preliminary evidence of synergy between panobinostat and TAK228, consistent with findings in other tumor types that mTOR inhibition in combination with panobinostat leads to improved cell killing (20, 21). We hypothesize that combinatorial regimen of TAK228, radiation, and panobinostat can effectively target H3.3 and H3.1K27M mutant cells in vitro and in vivo and that any mechanism of drug resistance are identifiable using cutting edge protein and gene studies. In this study, two Specific Aims will be tested: Specific Aim 1. Assess TAK228 efficacy in H3.3 and H3.1 DIPG models, alone and in combination with radiation and panobinostat.   Specific Aim 2. Determine mechanisms of cellular resistance to TAK228.  Our collaborative team has all necessary resources to carry the outlines experiments. The experiments detailed here will provide the pre-clinical justification of a phase I/II clinical trial of TAK228 in patients with newly diagnosed DIPG. 
 
Innovation. Our collaborative proposal is highly innovative in the following aspects: - Strong preliminary data indicating the efficacy of TAK228 for targeting DIPG tumor 

- Robust data indicating TAK228 administration more than doubles the median survival of mice bearing a well-accepted murine DIPG orthotopic model 

- Ability to target both mTORC1 and mTORC2 pathways using TAK228 

- mTORC1 inhibition by commonly used drugs (e.g. rapamycin and everolimus) often leads to upregulation of mTORC2, which may contribute to the relative lack of effectiveness of mTORC1 inhibitors. TAK228, which is a dual mTORC kinase inhibitor may have broader therapeutic use than rapalogs, due to its ability to inhibit both mTORC1 and mTORC2.

- Ability to inhibit DIPG growth and induce apoptosis at nanomolar concentrations.

- The agent is available for clinical trials in the United States through the CTEP program at NCI. 

- Collaborative proposal between two leading institutions in childhood brain tumor research.

- Access to robust and rare xenograft, allograft, and in vitro models of DIPG avenues.

- Potential to assess and identify combinatorial therapy of TAK228 for direct translational application of this agent. 

- Potential for identification of tumorigenic pathways and thus further allowing for novel therapeutic combinations. 
 
Feasibility. The proposed project is a highly feasible project as shown by our robust preliminary data and published manuscript (Cancer Letters, 2017). Our collaborative team of basic researchers and clinicians have the expertise and the resources to fully execute the proposed project.                                                     
 
Expertise. Dr. Nazarian (PI) at CNHS has extensive expertise studying the molecular profiling (mRNA, methylation, proteomics) of DIPGs. Dr. Raabe (Co-PI) a pediatric oncologist at Johns Hopkins University School has extensive experience in cell culture, molecular biology, immunochemical analysis and xenografting. Dr. Raabe’s laboratory had developed a primary DIPG line (JHH DIPG1) extensively used for preclinical testing across many laboratories.