Diffuse Intrinsic Pontine Gliomas

Introduction/Epidemiology

A diffuse intrinsic pontine glioma (DIPG), is a malignant tumor of the pons of the brainstem that originates in the supportive tissue called the glia. Specifically, these tumors are diffuse astrocytomas meaning that the tumor develops from astroglial cells of the brain stem. This tumor accounts for about 10%-20% of all brain tumors in children and roughly 80% of the 300-400 pediatric brainstem tumors that are diagnosed each year. The majority of children who develop this tumor type are between the ages of 5 and 10.

DIPGs are highly aggressive and result in median survival of less than a year and long term survival usually not exceeding 2 years (NIH National Cancer Institute). Symptoms of DIPG are referred to as the triad of symptoms which includes long tract signs (abnormal reflexes, hypertonia, motor deficits), ataxia (impaired coordination), and unilateral or bilateral cranial neuropathies (damage to the cranial nerves). More generalized symptoms include behavioral changes and decreases in school performance (Vanan and Eisenstat, 2015). Diagnosing this tumor consists of a combination of clinical findings and neuroimaging studies such as an MRI. Histological studies are not necessary to confirm the diagnosis, but it can be done for research purposes and for future therapies involving individual tumor profiling. At diagnosis, tumor grades can range from low-grade fibrillary astrocytomas (grade 2), to glioblastoma (grade 4). Postmortem evaluation usually reveals a grade 3 anaplastic astrocytoma or grade IV glioblastoma (NIH National Cancer Institute) .

Common Mutations

A variety of key mutations have been reported in postmortem evaluation of DIPGs as well as biopsies of patient tumors. Around 80% of DIPGs contain a mutation in either histone H3.1 (H3F3A) or H3.3 (HIST1H3B and HIST1H3C) genes suggesting that the H3 K27M mutation is likely a driver mutation for DIPG. H3K27M mutations have a co-occurrence rate of 75% for TP53. 40% for PDGFRA, 30% for ACVR1, and 22% for PPM1D. Additional mutations found in DIPGs include TP53 (55%), ACVR1 (24%), PDGFRA (16%), PPM1D (15%), ATRX/DAXX (9%), and MYCN/MYC (7.70%). H3K27M, ACVR1, PDGFRA, and PPM1D all cause epigenetic dysregulation which results in hypomethylation. The ATRX/DAXX mutation results in chromatin remodeling and MYCN/MYC mutations result in hypermethylation. These major mutations will be detailed in the following paragraphs (Long, Yi, Chen, Cao, Zhao, and Liu 2017).

Histone proteins are a major component of chromatin and histone H3, the histone protein mutated in 80% of DIPGs, exists in the 3 isoforms H3.1, H3.2, and H3.3. The N-terminal of these H3 proteins is an area that undergoes post translational modification such as methylation. Methylation status of the H3K27 plays a large role in the expression of cancer-related genes. EZH2 typically controls methyltransferase activity and thus the methylation status of H3K27M, while JMJD3/KDM6B and UTX/KDM6A control the de-methylation activities. However, it is known that about 80% of DIPGs harbour a mutation in an H3 variant which leads to the expression of H3K27M. This mutation exchanges the lysine residue at amino acid 27 for a methionine residue. The H3K27M mutation causes it to bind to the catalytic subunit of PCR2 (EZH2), and inhibit PCR2 function.

The result of this is an overall loss of H3K27me3 levels, which causes an alteration in the expression of several different genes (Long, et al., 2017). Induction of the H3K27M mutation in vivo demonstrated that the mutation was able to decrease the overall trimethylation status of H3K27 as well as increase the overall acetylation of H3K27. Again, 80% of DIPGs harbour the H3K27M mutation suggesting that it is a driver mutation in tumor development and that this specific amino acid substitution results in a gain of function mutation for that histone (Lewis et al., 2013). This is supported by a study done that tested every amino acid substitution at residue 27 of H3. Majority of the substitutions had minimal effects on the trimethylation levels of H3K27. Furthermore, several additional studies by the same researchers demonstrated the inhibitory effects of H3K27M mutation on PCR2 function. When lysine was replaced by methionine, the methionine side chain was able to interact with the aromatic residues of EZH2 active site (catalytic subunit of PCR2) and inhibit PCR2 function. This missense histone mutation proved to drastically alter gene expression because of this gain of function that leaves mutant H3 with the ability to to interact with the active site of several SET domain-containing methyltransferases. Though the H3K27M mutation is not present in many histones, global loss of H3K27me3 can be attributed to the mutation. Overall, this study suggests that this one missense mutation in the histone protein can globally disrupt positive feedback loops which leads to the inhibition of PCR2 and overall loss of H3K27me3 (Lewis et al., 2013).

Generally, DIPGs with an H3 mutation are considered more aggressive tumors and are associated with a lower overall survival rate. However, mutations in IDH1 can also affect the methylation status of H3K27. Mutant IDH1 can induce high levels of DNA hypermethylation by initiating CpG island methylator phenotype. This can cause an overall increase in H3K27me3 levels (Long, Yi, Chen, Cao, Zhao, and Liu 2017). Mutated IDH1 causes the accumulation of 2-hydroxyglutarate, which plays a role in epigenetic dysregulation and consequently hypermethylation that promotes an overall repression of several genes involved in anti-tumor responses. This suppresses the immune response and decreases T-cell infiltration of the tumor (Choy and Curry, 2017).

A smaller percentage of DIPGs harbour an ATRX loss of function mutation that has a 100% co-occurrence with H3K27M mutations of H3. ATRX-DAXX are subunits of the H3.3 chromatin remodeling complex at telomeres and other genomic sites. Mutations resulting in the loss of function of the ATRX-DAXX causes a reduction in the levels of H3.3 that are incorporated into heterochromatic regions. The reduction of H3.3 incorporation results in destabilization of telomeres which promotes the alternative lengthening of telomeres(Long, Yi, Chen, Cao, Zhao, and Liu 2017) .

Upregulation of the MYC pathway is also seen in a small number of DIPG patients. This occurs through the bromodomain and extraterminal (BET) family proteins that play a role in transcriptional activation through interactions with acetylated chromatin. Specifically, BET proteins play a role in the expression of certain oncogenes that are involved in cell cycle progression and apoptosis. Elevated levels of acetylated H3K27, which is associated with tumorigenesis, has been associated with increased levels of bromodomain containing proteins, BRD1 and BRD4. Increases in these proteins upregulate MYC, and increased MYC expression results in cell proliferation and increases a cell’s metastatic capacity (Long, Yi, Chen, Cao, Zhao, and Liu 2017)

ACVR1 mutations are found in nearly 25% of all DIPGs and have a co-occurrence with H3K27M mutation of 30%. The product of ACVR1 is ALK2 which is an activin receptor-like kinase-2 that is a member of the TGF-B family subgroup of type 1 bone morphogenetic protein (BMP) receptor. In addition to the role of BMP in endochondral skeleton growth and morphogenesis, evidence also suggests that BMP plays a large role in the differentiation and function of neural cells and tissues. ALK2 combines with type II BMP receptors to form tetrameric complexes which signal primarily through SMAD 1/5/8 BMP signaling. Normally, regulatory residues within ACVR1 function to suppress ALK2 activity by stabilizing its inactive form.

Mutations in these residues however increase basal canonical BMP Pathway signaling through SMAD. This gain of function mutation did not prove to have tumorigenic properties on its own however, and therefore requires the presence of a driver mutation to have tumorigenic effects. It was observed that ACVR1 mutants have the ability to increase cellular proliferation through SMAD signaling and the expression of the target genes ID1 and ID2 (Pacifica and Shore, 2015). ID1 and ID2 proteins that are both associated with actively proliferating cells and play a major role in many cancer types through their ability to promote cell survival, angiogenesis, and metastasis (Roschger and Cabrele, 2017). ID1 and ID2 expression are both also induced by the overexpression of H3K27M mutation. Co-expression of the ACVR1 mutant and H3K27M mutant result in an even greater overexpression of ID1 and ID2 and possible additive effects. The possible outcomes of ACVR1 mutations include cell proliferative and anti-apoptotic effects, deranged astroglial cell differentiation, cellular dedifferentiation, and cancer stem cell maintenance (Pacifica and Shore, 2015).

PDGFRA mutations occur in 16% of DIPGs and have a co-occurrence of 40% with H3K27M mutations (Long, Yi, Chen, Cao, Zhao, and Liu 2017). The PDGFRA is a receptor tyrosine kinase that is normally involved in cell migration, proliferation, and survival by signaling through PI3K/Akt, RAS/MAP kinase, Src kinase family, and PLC/PKC pathways. Each of these pathways have roles in tumorigenesis and therefore can play a major role in tumor development when PDGFRA is mutated and thus constitutively active (Paugh and et al., 2013).

Skipping the first treatment

Epigenetic Therapeutics

A large number of DIPGs contain epigenetic modifications in association with or not in association with gene mutations. These epigenetic alterations are therefore highly associated with DIPG development and progression. The global reduction of H3K27me3 levels is a major alteration that has become the target of epigenetic therapeutic strategies that target the enzymes that are responsible for the modifications of the chromatin. Inhibition of EZH2, the main

Increases in acetylation of H3K27 result in the opening of the chromatin and consequently the increase in transcriptional activity of that DNA. Transcription machinery is assembled there through the recruitment of BET proteins (BRD2, BRD3, and BRD4). These BET proteins have crucial roles in transcriptional activation and therefore also have oncogenic potential. This prominent role of BET proteins in transcriptional activity suggests that it could possibly be a viable target for therapies that inhibit BET activity. Small molecule BET inhibitors were developed and tested for their efficacy. JQ1 is a histone binding module inhibitor that binds competitively to bromodomains with high affinity and specificity. JQ1 is able to displace the binding of BRD4 to chromatin, which induces cell cycle arrest and apoptosis.

One major consequence of increased BET protein activity is the the overexpression of MYC/MYCN. Both medulloblastoma patients and mouse model-derived medulloblastoma cell lines where MYC was amplified were treated with JQ1. It was found that JQ1 was able to suppress MYC expression and MYC associated transcriptional activity which is notoriously difficult to target directly. This decreased overall cell viability in the medulloblastomas. BET inhibitors have also been tested for potential dual targeting by combining BET inhibitors such as JQ1 with the NOTCH-targeted gamma secretase inhibitor, MRK003 (Long, Yi, Chen, Cao, Zhao, and Liu 2017) . NOTCH overexpression has been implicated in a number of cancer types which has led to the development of therapies that interfere with NOTCH signaling. Research suggests NOTCH1 inhibition through MRK-003 treatment can reduce cell proliferation and also reduce B-catenin, MMP-2 and MMP-9 expression (Lai, Li, and Lin, 2017). This research suggests dual inhibition targeting BET proteins and NOTCH1 signaling may be an effective therapy, but it would require much more extensive research through preclinical trials and the translation into clinical trials (Long, Yi, Chen, Cao, Zhao, and Liu 2017).

Immunotherapy

Since the discovery of a lymphatic system within the CNS, immunotherapy has become a rapidly developing area of research for mechanisms of brain cancer metastasis and potential therapeutic treatments for brain cancers. It was found that about 50% of DIPGs express the mutation epidermal growth factor receptor variant III. The mutation allows EGFR to be constitutively active and ligand-independent. It has been demonstrated that cancer cells become somewhat dependent on the activation of these pathways and therefore inhibition of the stimulation of this pathway should decrease cell proliferation and survival. Anti-EGFRvIII vaccines are under currently being studied in clinical trials right now. Rindopepimut (CDX- 10) is an peptide vaccine that survived a phase I clinical trial that proved it was safed, tumor-specific, and immunogenic. It also demonstrated effectiveness in a multicenter phase II trial where it was combined with TMZ, an oral chemotherapeutic agent. However, CDX-110 did fail in a double blind randomized phase III trial. Research has continued though in working to produce an effective anti-EGFRvIII vaccine (An, et al., 2018).

In addition to EGFRvIII vaccinations, B7-H3 vaccines have also been proposed. B7-H3, also known as CD276, belongs to the immunoglobulin super family. It is a transmembrane glycoprotein that belongs to the B7-CD28 family and it has been implicated in the development of tumors (Long, Yi, Chen, Cao, Zhao, and Liu 2017). B7-H3 functions as an immune checkpoint and plays an important role in inhibiting T-cells. Overexpression of B7-H3 therefore contributes to the altered immune response and evasion of the immune system that is seen in highly malignant gliomas (Wang, 2018). Its presence in neuroepithelial tumors, but not neurons makes it tumor-specific antigen that is viable for targeting through vaccinations. Studies have demonstrated significantly high levels of B7-H3 present in DIPG, when compared to normal brain tissue, making it a potential therapeutic target (Long, Yi, Chen, Cao, Zhao, and Liu 2017). Other immune cell checkpoint molecules that work to suppress immune system activity and prevent immune overreaction have also been implicated in tumor development. Exploitation via upregulation of CTLA-4 or PD-1, immune system checkpoint proteins, can allow for cancer development by suppressing the immune system and evading anti-tumor immunity. This has led researchers to test mechanisms that block these two immune checkpoints. This method has shown promising results in several malignancies. The humanized antibodies pidilizumab and pembrolizumab, which target PD-1, are both under clinical investigation for DIPG specifically. Pidilizumab is in a phase I/II clinical trial , but studies of pembrolizumab have been suspended due to widespread adverse reactions. Much further testing of immune checkpoint inhibitors must occur to evaluate the safety and efficacy of using these immune checkpoint inhibitors (Long, Yi, Chen, Cao, Zhao, and Liu 2017).

Research at Stanford University School of Medicine has demonstrated the ability to engineer chimeric antigen receptor T-cells (CAR-T cells) to target and destroy nearly all cancer cells in a mouse model. This is the first mouse model study that has successfully eliminated the entire diffuse intrinsic pontine glioma. The first step in developing these CAR-T cells involved identifying tumor cell surface markers that could be used as targets for the engineered T cells. The idea is that some of the patient’s T cells can be removed from their body, engineered to attack a cell surface antigen that is tumor-specific, and then the T cells are re-inserted back into the patient with the hope that the T cells will recognize the antigen and destroy the cancer cell. The research team at Stanford identified a sugar molecule called GD2 that was present in high numbers on the surface of DIPG tumors. The overexpression of GD2 is a result of the H3K27M mutation that is seen in up to 80% of DIPG cases. This mutation and the excess production of GD2 is responsible for driving the growth of the tumor. The research team designed anti-GD2 CAR-T cells that in culture, destroyed all DIPG cells harbouring the K3K27M mutation.

The GD2 CAR-T cells were also tested in mice whose brainstem was implanted with human DIPG tumors. 7-8 weeks after the tumor was established, each mouse received an intravenous injection of the engineered T cells that were either GD2 specific, or specific for a different target which served as a control. Mice that were the recipient of the GD2-CAR T cells experienced had an undetectable tumor after only 14 days. After 50 days, all of the mice were euthanized and then examined using immunostaining. The mice treated with the GD2-CAR T cells had just a few dozen cancer cells remaining whereas the control treatment mice had tens of thousands of cells remaining. The cells that were remaining in the GD2-CAR T cell treated mice did not express GD2, which is a cause of concern for cancer recurrence. As with most treatments in this delicate and critical area of the brain, there are risks associated with the inflammatory response that is induced by the T cells. Additionally, this form of immunotherapy did successfully clear out majority of the DIPG tumor cells, but those cells that were not cleared man not susceptible to immunotherapy. This just further demonstrates the idea that there is likely no one treatment that can clear out DIPG tumor cells, but rather that it will take a combination of many therapy types to completely clear the tumor. This approach will now be introduced into human trials (Mount, et al., 2018).

Nanoparticle Delivery Systems

Major barriers exist when attempting to precisely target and deliver chemotherapeutic agents directly at the tumor site. Issues such as penetrating the highly selective BBB, minimizing toxic effects to surrounding healthy cells, and achieving even intratumoral distribution. A new drug delivery system is currently being studied that uses genetically engineered stem cells that contain nanoparticle delivery systems. Traditional glioma gene therapy utilizes viral vectors to transport the genes to cell carriers. However, this method comes with the potential for infection related cell damage and issues with immune response. Nanoparticles that are bound to DNA however, avoid these potential risks which has opened the gate for use of stem cells as carriers. Researchers have demonstrated the efficacy of this through their successful gene delivery of polyethylenimine (PEI)-DNA coated silica nanoparticles into hMSCs. 75% of the hMSCs took up the particles delivered. Additionally, the hVEGF gene was successfully delivered into hMSCs using biodegradable poly beta-amino ester nanoparticles. The delivery of the hVEGF gene did enhance the production of VEGF by the target cells. The limit to these two studies is that they were not specifically testing the efficacy of this in regards to glioma treatment. However, they have provided researchers with models that effectively demonstrate the potential use of these nanoparticle delivery systems in glioma treatment (Long, Yi, Chen, Cao, Zhao, and Liu 2017).

Small Molecule Inhibitors

Maternal embryonic leucine zipper kinase (MELK) encodes is a serine/threonine protein kinase that is known to be expressed in early embryonic cellular stages. It has now also been proven to be expressed in a wide variety of adult tissues too, such as the thymus and spleen. MELK expression was also found in in T cell lineages, macrophages, and monocytes, but not B cell lineages. An immune complex kinase assay demonstrated that MELK has active kinase catalytic activity, which suggests that there is a functional gene product in these tissues. This led researchers to believe that MELK plays a role in the signal transduction of specific lineages of hematopoietic cells. Further studies of MELK have been able to uncover additional functions of MELK and mechanisms of signal transduction. The strong role of MELK in proliferating cells during early embryonic development and the high levels of expression MELK in cancer cells has led researchers to look into MELK’s role in cell cycle regulation. In an intestinal cancer cell line, MELK expression patterns were similar to that of cyclin A, cyclin B, and CDK4, which are all known cell cycle regulators. Brain tumor expression of MELK mRNA exhibited similar expression levels to known mitosis-phase regulatory proteins. In other words, MELK expression was similar to that of other known kinases and enzymes that are involved in cell cycle regulation and progression. Further studies on MELK suggested that it likely has a prominent role in cell cycle regulation and may be required for cell cycle progression. Additionally, MELK’s role in apoptotic pathways and anti-apoptotic pathways are unclear. In colon cancer cells, overexpression of MELK results in the overexpression of p53 and it exhibits a pro-apoptotic function. In contrast, glioblastoma multiforme cancer cells have an inverse relationship with MELK. Overexpression of MELK induced silencing of p53,

Work Cited

1. https://www.cancer.gov/types/brain/hp/child-glioma-treatment-pdq#_1
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617108/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5525007/
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951439/
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753137/
6. https://biosignaling.biomedcentral.com/articles/10.1186/s12964-016-0161-y
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3800209/
8. https://www.spandidos-publications.com/mmr/17/2/2472
9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860944/
10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669623/
11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214371/"

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