The ChadTough Defeat DIPG Foundation is excited to have hosted a Q and A session with leading brain cancer experts who shared information about current research in the DIPG field, as well as some upcoming trials you can expect to see this year.

DIPG Biology & Histology

DIPG: Pathophysiology and Histology

Pediatric diffuse intrinsic pontine gliomas (DIPGs) are brain tumors that originate in the pons (the middle portion of the brainstem), and grow throughout (“infiltrate”) the pons, taking up at least half of this vital structure.  They can grow into other area of the brainstem, and grow into the CSF-filled fourth ventricle next to the pons.  DIPGs are tumors of astrocytic (or glial) cells, which are supportive cells of the brain named for their star-like shape.  All gliomas, including DIPGs, are given grade I to IV, based on the presence of aggressive histologic features.  Interestingly, DIPGs can be any grade, and their behavior and prognosis is more closely linked to molecular features, such as the presence of an H3 K27M mutation (described in more detail below). Recent multi-institutional sequencing efforts and pre-clinical models have greatly advanced our understanding of the biology driving DIPG formation and growth.

DIPGs are classic examples of developmentally-based tumors.  In the cell division necessary to build and maintain the pons during infancy and childhood, errors can occur during DNA replication.  In our current understanding, the first steps of a DIPG occur when mutations occur in astrocyte precursor cells within the pons.  The overwhelming majority of these mutations are inconsequential or will be repaired by cell machinery.  However, if just the wrong error occurs, such as a mutation leading to an amino acid substitution within the histone tail at position K27 of the gene H3F3A (H3.3) or HIST1H3B (H3.1), the cell with mutated DNA will undergo a critical change in behavior.1,2

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H3 K27M Mutation: Early Tumorigenesis and Therapeutic Targeting

With a H3K27M mutation in the correct developmental environment, such as the brainstem or thalamus of an infant or child, the astrocyte precursor cell will divide more frequently and begin expressing genes important for early tumor growth.1-3  About 80% of DIPGs carry this mutation as an early, or clonal, event.4  Alterations in other genes, such as MYCN, can drive the formation of a DIPG without a histone mutation.4  Other tumor-driving mutations co-occur as clonal mutations, in part driven by the excess proliferation of these early tumor-forming cells. Some of these early driving mutations are seen in non-brainstem high-grade gliomas (HGGs) of adults (i.e., TP53 or PIK3CA) while others are unique to DIPG (i.e., ACVR1).  Overall, DIPGs carry less genetic alterations than adult high-grade glioma and are more likely to carry the same driving mutations throughout the tumor (“spatially homogenous”).5 H3 K27M-mutant cells generally carry additional concurrent genetic alterations, which are likely necessary to result in the full invasive phenotype of DIPG.6

While the two histone variants (H3.3 and H3.1) are nearly identical in sequence, histone H3.3 (encoded by H3F3A) is incorporated into nucleosomes in a manner that is replication-independent, while H3.1 (encoded by HIST1H3B) is only incorporated during DNA synthesis.7 Interestingly, the histone gene that the K27M mutation occurs in results in distinct tumor biology in terms of prognosis, concurrent mutations, location and age.  H3.3 K27M is associated with the worst overall survival, followed by H3.1, and then histone wild-type status.8  The K27M mutation is believed to contribute to oncogenesis by interfering with normal post-translational modifications of the histone H3 protein.9

Among the histone post-translational modifications, the H3 tail lysine 27 residue plays a pivotal role in the overall functional properties of DNA, and acetylation and methylation results in significant downstream impacts. It is perhaps not surprising that the H3K27M mutation results in significant dysfunction of normal epigenetic signaling and programming at the H3K27 locus.10 Heterozygous (gain of function) H3K27M mutations in either the H3F3A or HIST1H3B gene have been shown to suppress EZH2, a catalytic subunit of polycomb repressive complex 2 (PRC2) which trimethylates the histone H3 at lysine 27. H3K27M inhibition of PRC2 causes a global reduction in total chromatin H3 dimethylation (K27me2) and trimethylation (K27me3). This global loss of H3K27 methylation results in both increased cell proliferation potential and decreased differentiation ability.10

Spatially and temporally, DIPG and H3K27M DIPGs are felt to be relatively homogenous in terms of primary genetic drivers.11,12 However, a recent study employing single-cell RNA sequencing (scRNA-seq) on thousands of individual cells from six H3K27M primary gliomas has given us new insight into the cellular and transcriptional architecture of H3K27M tumors.11 H3K27M gliomas contain a majority of oligodendrocyte precursor cells (OPC) which remain in a state of rapid proliferation, fueling tumor growth and sustained by PDGFRA signaling.11

Histone deacetylases (HDAC) are responsible for the removal of acetyl groups from histone tails. HDAC inhibitors induce epigenetic changes, primarily increases in H3 acetylation, which reduce cellular proliferation and viability in certain tumor cells.13  A study by Grasso et al showed that treatment with the HDAC inhibitor panobinostat restored H3 acetylation of K27M-mutant DIPG cell lines, and was also efficacious in mouse DIPG xenografts.12 However, a more recent study demonstrated that the activity of HDAC inhibitors is similar among wild-type and K27M-mutated DIPG cell lines and the inhibitors had little to no efficacy in in vivo studies.13 Results of clinical trials utilizing HDAC inhibitors in pediatric HGG and DIPG have had suboptimal outcomes, potentially due to the poor blood brain barrier (BBB) permeability of these agents and/or secondary to efflux transport.14-19 However, identification of populations that may specifically benefit from HDAC inhibitor therapy or using concurrent therapies to improve BBB penetration may improve outcomes.20

ONC201 is an investigational molecule that antagonizes dopamine receptor D2 (DRD2) and was first identified in a screen for compounds that induce the cytokine TNF-related apoptosis-inducing ligand (TRAIL).21,22 ONC201 was reported to inactivate kinases Akt and ERK which leads to Foxo3a-driven upregulation of TRAIL in the nucleus and promotes cancer cell death through activation of the integrated stress response.21,23 ONC201 has shown promise in the clinical setting as an anti-tumor agent with high CNS penetration and is currently being investigated in clinical trials for adult and pediatric H3K27M glioma, including one expanded access protocol.24-27

In a patient-derived cell viability panel, ONC201 potency was highest against H3K27M-mutant glioma with reduced activity against wildtype glioma cell lines.26 ONC201 clinical efficacy in adult H3K27M-mutant glioma was first demonstrated in a 22-year-old patient who experienced durable 96% objective response.25,26 Furthermore, significant clinical and radiographic response has been reported in a DIPG patient treated with ONC201.28 Based on this early promising data, Oncoceutics (the manufacturer of ONC201), is applying for accelerated FDA approval, which if granted, would be the first agent with that status for H3K27M-mutant glioma. Multiple phase 2 trials with ONC201 are being developed for H3K27M-mutant glioma through the Children’s Oncology Group (COG) and the Pacific Neuro-Oncology Consortium (PNOC).


RTK-RAS-RAF-PI3K pathway

Through whole genome and RNA sequencing, Wu and colleagues found that mutations affecting RAS-RAF-PI3K signaling were present in 69% of DIPG.4 According to a large meta-analysis of 1,000 pediatric HGG and DIPGs, Mackey et al found that mutations affecting the various components of the RAS-RAF-PI3K signaling pathway tend to co-segregate with specific histone mutations: alterations impacting PDGFRA with H3F3A K27M, alterations impacting PI3K/mTOR with HIST1H3B K27M, and alterations impacting ERK (predominantly BRAF mutations) with histone wild-type tumors.8  Mutations and amplifications of RTKs, such as PDGFRA and PDGFRB, or their ligands, PDGFA and PDGFB, result in activation of both the PI3K and the RAS/RAF pathways. The PI3K pathway may also be activated through other somatic variations such as PTEN deletion or PIK3CA mutations.8,29 In the recent study published by Mackay et al, there were a total of 37 pediatric HGG cases with HIST1H3B K27M, 14 of which also possessed mutations in either PIK3CA or PIK3R1.8



Through the use of whole-genome sequencing with methylation, expression, and copy number profiling, mutations in the activin A receptor type 1 gene (ACVR1) were found in about 20% of DIPGs.30 ACVR1 mutations are typically found in concurrence with a HIST1H3B K27M mutation and in the absence of TP53 mutation in DIPGs.30,31 Mutations of the ACVR1 gene are the most common genetic abnormalities in DIPG, after H3F3A and TP53 mutation.30 DIPG cases with ACVR1 mutations have increased levels of phosphorylation of SMAD1 and SMAD5.30 In vitro modeling also suggests that mutations in ACVR1 result in constitutive activity of the receptor, leading to increased expression of activin signaling targets, including ID1 and ID2.30 Germline mutation of ACVR1 can produce the autosomal dominant disorder fibrodysplasia ossificans progressiva (FOP), including several specific point mutations that have also been seen in DIPG. It is as of yet unclear exactly how these mutations may contribute to tumor growth, but it seems that they are not sufficient to drive oncogenesis as FOP patients do not have a propensity to develop tumors.32

Clinically Integrated Sequencing Precision Medicine for DIPG

Four recent studies demonstrated the feasibility and utility of clinical integrated sequencing in pediatric brain tumor patients, including DIPG.23,33-35  In part driven by the lack of efficacious adjuvant therapies for children and young adults with DIPG, therapies targeted to the unique biology of each tumor have increasingly replaced traditional cytotoxic chemotherapies on and off clinical trial.  While this field is still in its infancy, there is substantial promise to the incorporation of precision medicine into the management of children with DIPG.


Therapeutic monitoring and the utility of liquid biopsies

As therapeutic decisions are guided by changes in tumor growth, it is of paramount importance that an accurate and minimally-invasive method be developed to monitor treatment response. Current research is being done to assess whether liquid biopsy for H3K27M is a feasible means by which to do so.

Brain tumor cells release circulating tumor DNA (ctDNA) into surrounding cerebrospinal fluid (CSF) and can cross the BBB into plasma.36 This cellular mechanism has important clinical utility for diagnostic tests, genetic profiling of tumors, and therapeutic monitoring. ctDNA has already been used to track disease progression in adult cancers including liver, lung, and breast cancers; the application of this tool to the pediatric CNS population is presently underway.

Digital droplet PCR (ddPCR) is an ultrasensitive PCR method allowing for detection and quantification of ctDNA at extremely low concentrations. PCR reactions are partitioned into thousands of droplets, allowing for highly sensitive and rapid quantification of low mutant allele frequencies (MAF). As H3K27M is the most frequent mutation in pediatric brainstem gliomas and is closely associated with clinical outcomes, it has been the subject of much ddPCR experimentation.

Sequencing of ctDNA in CSF also allows investigators to detect tumor mutations 37. The advantage that sequencing has over ddPCR is the ability to detect innumerable mutations at once, including but not limited to H3K27M, with high sensitivity and specificity. The use of sequencing to diagnose and monitor treatment response of tumor genetics in CSF is ongoing and holds great promise.

Mutant H3K27M copies from CSF of pediatric DIPG patients is positively correlated with contrast-enhancing cross-sectional tumor area on MRI.38 Further, a decrease of H3K27M MAF by both ddPCR analysis and sequencing was observed following radiotherapy treatment. The ddPCR and sequencing results correlated with a decrease in tumor burden as measured by MRI tumor volume.39

These preliminary data demonstrate the feasibility and promise of liquid biopsies to monitor treatment response in pediatric DIPG patients. Current barriers to wider spread use include adoption of serial lumbar puncture as standard of care for surveillance of DIPG and the expansion of CLIA-certified labs offering liquid ctDNA analysis.


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  2. Khuong-Quang DA, Buczkowicz P, Rakopoulos P, et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol. 2012; 124(3):439-447.
  3. Sturm D, Witt H, Hovestadt V, et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell. 2012; 22(4):425-437.
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  5. Hoffman LM, DeWire M, Ryall S, et al. Spatial genomic heterogeneity in diffuse intrinsic pontine and midline high-grade glioma: implications for diagnostic biopsy and targeted therapeutics. Acta neuropathologica communications. 2016; 4:1.
  6. Pathania M, De Jay N, Maestro N, et al. H3.3(K27M) Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas. Cancer Cell. 2017; 32(5):684-700 e689.
  7. Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell. 2004; 116(1):51-61.
  8. Mackay A, Burford A, Carvalho D, et al. Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma. Cancer Cell. 2017.
  9. Lewis PW, Muller MM, Koletsky MS, et al. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science. 2013; 340(6134):857-861.
  10. Wierzbicki K, Ravi K, Franson A, et al. Targeting and Therapeutic Monitoring of H3K27M-Mutant Glioma. Current Oncology Reports. 2020; 22(2):19.
  11. Filbin MG, Tirosh I, Hovestadt V, et al. Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq. Science. 2018; 360(6386):331-335.
  12. Grasso CS, Tang Y, Truffaux N, et al. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nature medicine. 2015; 21(6):555-559.
  13. Hennika T, Hu G, Olaciregui NG, et al. Pre-Clinical Study of Panobinostat in Xenograft and Genetically Engineered Murine Diffuse Intrinsic Pontine Glioma Models. PLoS One. 2017; 12(1):e0169485.
  14. Muscal JA, Thompson PA, Horton TM, et al. A phase I trial of vorinostat and bortezomib in children with refractory or recurrent solid tumors: a Children’s Oncology Group phase I consortium study (ADVL0916). Pediatr Blood Cancer. 2013; 60(3):390-395.
  15. Hummel TR, Wagner L, Ahern C, et al. A pediatric phase 1 trial of vorinostat and temozolomide in relapsed or refractory primary brain or spinal cord tumors: a Children’s Oncology Group phase 1 consortium study. Pediatr Blood Cancer. 2013; 60(9):1452-1457.
  16. Fouladi M, Park JR, Stewart CF, et al. Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children’s Oncology Group phase I consortium report. J Clin Oncol. 2010; 28(22):3623-3629.
  17. Rasmussen TA, Tolstrup M, Moller HJ, et al. Activation of latent human immunodeficiency virus by the histone deacetylase inhibitor panobinostat: a pilot study to assess effects on the central nervous system. Open Forum Infect Dis. 2015; 2(1):ofv037.
  18. Wang C, Eessalu TE, Barth VN, et al. Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain. Am J Nucl Med Mol Imaging. 2013; 4(1):29-38.
  19. Hooker JM, Kim SW, Alexoff D, et al. Histone deacetylase inhibitor, MS-275, exhibits poor brain penetration: PK studies of [C]MS-275 using Positron Emission Tomography. ACS Chem Neurosci. 2010; 1(1):65-73.
  20. Marini BL, Benitez LL, Zureick AH, et al. Blood-brain barrier-adapted precision medicine therapy for pediatric brain tumors. Transl Res. 2017; 188:27 e21-27 e14.
  21. Allen JE, Kline CL, Prabhu VV, et al. Discovery and clinical introduction of first-in-class imipridone ONC201. Oncotarget. 2016; 7(45):74380-74392.
  22. Allen JE, Krigsfeld G, Mayes PA, et al. Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects. Science translational medicine. 2013; 5(171):171ra117.
  23. Kline CN, Joseph NM, Grenert JP, et al. Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy. Neuro-oncology. 2017; 19(5):699-709.
  24. Arrillaga-Romany I, Chi AS, Allen JE, Oster W, Wen PY, Batchelor TT. A phase 2 study of the first imipridone ONC201, a selective DRD2 antagonist for oncology, administered every three weeks in recurrent glioblastoma. Oncotarget. 2017; 8(45):79298-79304.
  25. Arrillaga-Romany I, Odia Y, Prabhu VV, et al. Biological activity of weekly ONC201 in adult recurrent glioblastoma patients. Neuro-oncology. 2020; 22(1):94-102.
  26. Chi AS, Tarapore RS, Hall MD, et al. Pediatric and adult H3 K27M-mutant diffuse midline glioma treated with the selective DRD2 antagonist ONC201. Journal of neuro-oncology. 2019:1-9.
  27. Hall MD, Odia Y, Allen JE, et al. First clinical experience with DRD2/3 antagonist ONC201 in H3 K27M–mutant pediatric diffuse intrinsic pontine glioma: a case report. Journal of Neurosurgery: Pediatrics. 2019; 23(6):719-725.
  28. Hall MD, Odia Y, Allen JE, et al. First clinical experience with DRD2/3 antagonist ONC201 in H3 K27M–mutant pediatric diffuse intrinsic pontine glioma: a case report. Journal of Neurosurgery: Pediatrics. 2019; 1(aop):1-7.
  29. Koschmann C, Farooqui Z, Kasaian K, et al. Multi-focal sequencing of a diffuse intrinsic pontine glioma establishes PTEN loss as an early event. npj Precision Oncology. 2017; 1(1):32.
  30. Buczkowicz P, Hoeman C, Rakopoulos P, et al. Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nature genetics. 2014; 46(5):451-456.
  31. Fontebasso AM, Papillon-Cavanagh S, Schwartzentruber J, et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nature genetics. 2014; 46(5):462-466.
  32. Taylor KR, Vinci M, Bullock AN, Jones C. ACVR1 mutations in DIPG: lessons learned from FOP. Cancer Res. 2014; 74(17):4565-4570.
  33. Ramkissoon SH, Bandopadhayay P, Hwang J, et al. Clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors. Neuro-oncology. 2017; 19(7):986-996.
  34. Koschmann C, Wu Y-M, Kumar-Sinha C, et al. Clinically Integrated Sequencing Alters Therapy in Children and Young Adults With High-Risk Glial Brain Tumors. JCO Precision Oncology. 2018(2):1-34.
  35. Cole BL, Lockwood CM, Stasi S, et al. Year 1 in the Molecular Era of Pediatric Brain Tumor Diagnosis: Application of Universal Clinical Targeted Sequencing in an Unselected Cohort of Children. JCO Precision Oncology. 2018(2):1-13.
  36. Wang Y, Springer S, Zhang M, et al. Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci U S A. 2015; 112(31):9704-9709.
  37. Miller AM, Shah RH, Pentsova EI, et al. Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid. Nature. 2019; 565(7741):654-658.
  38. Stallard S, Savelieff MG, Wierzbicki K, et al. CSF H3F3A K27M circulating tumor DNA copy number quantifies tumor growth and in vitro treatment response. Acta Neuropathol Commun. Vol 6. England2018:80.
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