Recently Reviewed Research
1. Nuclear Factor-kappa B (NF-kappa B) / Leucine Zipper down regulated in cancer 1 (LDOC1)
NF-kappa B pathway is thought to regulate the expression of interleukin 6 (IL-6) in posterior fossa group A ependymoma 1. It is thought that IL-6 secretion in posterior fossa group A ependymoma drives an immune suppressive phenotype which polarizes infiltrating monocytes. Based on work from Geisinger, et al, knockdown of NF-kappa B led to down regulation of IL-6 in an vitro cell culture. As part of this study, another gene called Leucine Zipper down regulated in cancer 1 (LDOC1), which is known to be a transcriptional repressor of NF-kappa B, was found to be inactivated by hypermethylation and had decreased expression in posterior fossa group A ependymoma cell lines. A drug called 5-AZA-DC, which is a non-selective demethylator, was then used which caused upregulated LDOC1 expression and a consequent decrease in IL-6 secretion.
Comment: Use of this pathway as a therapeutic target would depend entirely on identifying a demethylating agent that would selectively target LDOC-1 in ependymoma cells. The idea has not been documented to be tested in any experimental model except cell culture. The whole-body effects of a DNA de-methylating agent are unknown.
2. Chimeric Antigen Receptor (CAR) T cell immunotherapy
This type of therapy which is already approved for several types of leukemia involves modifying a patient's existing T cells and adding a receptor which is found on the surface of the tumor cell. Seattle Children's hospital is already conducting a phase I trial with ependymoma utilizing the HER2 (ERBB2/receptor tyrosine-protein kinase) receptor, which is a receptor also found in breast cancer.
Comment: The effectiveness of this type of therapy will depend on finding a robust marker present only on the ependymoma tumor cells and on the degree to which the ensuing inflammatory response will be tolerated by the patient. A search for other markers would be a worthy effort as the the HER2 receptor will only likely be present on a subset of ependymoma patients. 2
3. Captopril
There is some weak evidence from a 2001 article 3 that the commonly used antihypertensive drug captopril may mitigate the migration and adhesion abilities of ependymoma. There was a trial performed by Rooprai at al in 2001 where this drug was tested on an ependymoma derived cell line.
Comment: Based on a literature review, it does not appear that this hypothesis was tested again in follow-up testing. Captopril is noted to cross the blood pain barrier. If there were some utility with this it could potentially be useful as an adjuvant therapy.
4. Immunotherapy with adV-tk
There was a recently published phase I trial authored by Kieran, et al, 4 that investigated the use of gene mediated cytotoxic immunotherapy (GMCI) where aglastimagene besadenovec (Adv-tk) was injected directly into the tumor bed after resection followed by an anti-herpetic prodrug. This approach leads to the local creation of nucleotide analogs that result in cell death and the release of tumor neoantigens converting "cold" tumor microenvironment into "hot" microenvironment which stimulates the immune system through the activation of the STING (stimulate of interferon genes) pathway resulting in cytokine production and super antigen mediated T-cell activation. There were only 8 patients in this trial and one with a recurrent supratentorial ependymoma (WHO II) with two prior surgeries. The ependymoma patient was still alive 47 months after treatment. Patients with ependymoma involving the brainstem were excluded from the trial. Prior to this trial, this technique had only been tried in adults.
Comment: This study did demonstrate some degree of efficacy in a number of the patients involved. It is interesting it was limited to ependymomas located in the suptratentorial location only suggesting some uncertainty of how adjacent eloquent structures in the brainstem would fare with the collateral inflammation that would likely result from this approach. The authors of this study do mention a possible phase II trial that is currently under design.
5. CXorf67 and posterior fossa type a ependymoma (PFA)
Based on recent research by Pajtler, et al 5 evaluating 675 PFA tumors, PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27 - me3) and missense mutations in a gene named CXorf67 (which presumably causes over expression of this gene). Immunopreciptation/mass spectrometry experiments by Pajtler, et al detected EZH2, SUZ12 and EED proteins, which are components of PRC2 (protein ribosomal complex 2), bound to the product of a gene called CXorf67 with no consequent expression of H3 K27 - me3 in Daoy cells (a medulloblastoma cell line). Enforced reduction of CXorf67 in Daoy cells restored H3 K27 - me3 levels to normal. An additional experiment enforcing expression of CXorf67 in HEK293T (a human cell line, derived from the HEK 293 cell line, that expresses a mutant version of the SV40 large T antigen.) and neural stem cells also reduced H3 K27 - me3 levels. A follow up article published by Hubner, et al (6), performed mass spectrometry and peptide modeling analysis to identify the functional domain of CXorf67 responsible for binding and inhibition of the protein EZH2. They found that a highly conserved peptide sequence located at the c terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain of EZH2. This interaction blocks EZH2 methytransferase activity and inhibits PRC2 function causing de-repression of PRC2 target genes including genes involved in neurodevelopment. The authors conclude that expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking k27M mutated histones. Based on its function, CXorf67 has been renamed to EZH Inhibitory Protein (EZHIP).
Comment: Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67.
6. Amblyomin-X
Amblyomin-X is a Kunitz type molecule which inhibits the function of protein degrading enzymes by specifically interacting with the kunitz domain of these protease molecules inactivating them. This particular compound was isolated from the salivary glands of a tick found in Brazil named Amblyomma cajennense. It has possible antiangiogenic and anticoagulant and anti-tumor properties as noted in a recent article by Pavon, et al7. Pavon et al, utilized a model of pediatric posterior fossa anaplastic ependymoma in primary culture and utilizing a intracranial xenograft model. The models were based on 4 primary cultures derived from patients with pediatric posterior fossa ependymoma, group A (anaplastic, WHO Grade III). Endings in this study suggested that Amblyomin-X was able to induce cell death in a more significant percentage of ependymome cells compared to cisplatin. The drug showed some specificity for tumor cells over a control of human amniotic fluid stem cells (hAFSC). A variety of techniques were utilized to assess tumor burden in the culture and animal models including MRI and histological analysis of tumor tissues.
Comment: The study by Pavon, et al, is poorly written with a complex methodology. The validation of the tumor model is a potential weakness here as it has been demonstrated elsewhere in cell culture that the epigenetic of a tumor can change once it has been removed from its native environment. The mouse tumor model also is suspect as the main confirmation of the identity of the transplanted tumor was mainly histological. That being said, the findings do appear to suggest some specificity for the cells in the ependymoma models used. This compound does warrant additional investigation.
8. Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma.
Dobson et al, J Immunol 2009; 183:7428-7440 8
In this study, tumor gene expression microarray profiles from pediatric ependymoma clinical samples obtained prior to standard treatment with surgery and radiation were subjected to ontological analyses with the aim of identifying outcome-associated biological factors. Ontology analyses revealed that genes associated with non-recurrent ependymoma were predominantly immune function related. Additionally, increased expression of immune related genes was correlated with longer time to progression in recurrent ependymoma. Histological analysis of a subset of these immune function genes revealed that their expression was restricted to a subpopulation of tumor infiltrating cells. Analysis of tumor infiltration immune cells showed increased infiltration of CD4+ T cells in the nonrecurrent ependymomas. No genomic sequences for SV40, BK, JC or Merkel polyoma viruses were found in the non recurrent ependymoma.
Comment: The authors hypothetically suggest that anti-tumor immune responses after surgery and radiation may shift the balance of equilibrium in favor of the host by increasing the immune/tumor ratio resulting in elimination of residual tumor by the immune system resulting in a more favorable outcome for the patient. Curiously, the presence of the SV40, BK, JC and Merkel polyoma viruses were not detected in non recurrent cases of ependymoma, indicating that these four viruses do not play a role in triggering an ongoing immune response to the presence of tumor. The authors do add that some other tumor not tested for may be responsible for this. A weakness of this presentation is that different types of ependymomas were included in the study and a relatively small number of cases were included. Recently, ependymoma has become viewed as a vast spectrum of diseases with different behaviors and response to treatment only unified by the fact that they have a similar histological appearance but share little else in terms of their molecular biology. The only useful clinical possibility that can be deemed from this investigation is that some tumor types may potentially be managed by stimulation of the native immune system.
8. TET2 promotor methylation and TET2 protein expression in pediatric posterior fossa ependymoma.
Pierscianek, et al, Neuropathology. 2019 Nov 28. doi: 10.1111/neup.12615.
Pediatric posterior fossa ependymoma (PF) is associated with a hypermethylated phenotype. The ten-eleven translocation methylcytosine dioxygenase 2 (TET2) gene (TET2) has been linked to the regulation of DNA methylation. TET2 promotor methylation and protein expression was analyzed in this study to assess the role of TET2 in PF. Medical records of all PF cases at the authors institution were evaluated between 1993 and 2015 regarding tumor histology, grade, tumor location, gender, age, tumor recurrence, distant metastasis, survival and time to progression. TET2 promotor methylation using methylation-specific polymerase chain reaction was then evaluated in each identified case. 37 cases were identified for inclusion into the study. TET2 protein expression was assessed using immunohistochemistry. Low TET2 expression was detected in seven of 17 cases. The authors found an association between low TET2 expression and tumor recurrence (P = 0.049). A TET2 promotor methylation was detected in five of 10 cases. There was no association between the TET2 promotor methylation with recurrence, tumor grade or gender. TET2 promotor methylation and low TET2 expression was detected in a subgroup of PF. The authors felt their data showed an association between low TET2 expression and tumor recurrence in PF.
Commentary: The TET family of enzymes promote demethylation. Decreased activity of this enzyme in glioblastoma has been associated with DNA hypermethylation. This article suggests, based on a review of a relatively small number of patients that low TET2 expression may contribute to hypermethylation and appears to correlate with tumor recurrence in posterior fossa disease. TET2 would likely not make a good drug target as it is an enzyme widely used by normal cells and selectively activating this gene would have unclear consequences. Assessing expression of this gene may be most useful as a marker of disease that is more likely to be recurrent.
9. Childhood cerebellar tumours mirror conserved fetal transcriptional programs.
Maria Vladoiu, et al, Nature. 2019 Aug;572(7767):67-73. doi: 10.1038/s41586-019-1158-7. Epub 2019 May 1
The study utilized large scale single cell RNA sequencing (scRNA-seq) to develop a 'cellular scaffold' for cerebellar development in which the transcriptomes of distinct cellular hierarchies can be determined at various points in time in a mouse model and compared to the transcriptomes of childhood cerebellar tumors. The comparison between the two transcriptomes was then used to identify a lineage restricted population of cells in the cerebellum that most closely mirrored the transcriptome of the tumor of interest. It was thought that due to the low mutational burden noted in many cerebellar tumors, it was likely that the tumor would be similar to its cell of origin. The hope with this evaluation is to discover developmental checkpoints that are defective in cerebellar tumors to ascertain tumor related changes in transcriptional programs. Insights into the process of transformation from a early cerebellar developmental cell to a tumor cell could potentially identify novel therapies. Using this evaluation, it was found that posterior fossa A (PFA), and posterior fossa B (PFB) ependymomas are transcriptionally most similar to the gliogenic progenitor 1 cell cluster, the proliferating ventricular zone progenitors and to a new cluster of cells called the 'roof plate like' stem cells. This finding is consistent with prior findings that PFA and PFB be classified as gliomas arising from radial glial cells. More specifically, these authors found a specific match to the gliogenic progenitor cells present on embryonic day 16. The authors also found that PFA tumors are unique amongst the childhood cerebellar tumors in that they lack more differentiated cell types. The authors argues that this lack of differentiation suggests the likely presence of a differentiation block in PFA ependymomas. Lastly, the authors relate that the normal, non transformed cell populations that transcriptionally best match cerebellar tumors are only present in utero or immediately post-natally.
Commentary: The main limitation of this evaluation is the use of a mouse model for comparison. Performing the same evaluation using human tissue as a comparison would have been more time consuming and difficult. The conclusion here that PFA/PFA originate from distinct populations of immature cerebellar cells is fascinating but not entirely unexpected as the size of many of these tumors at presentation indicates that that they were present long before clinical detection and likely present during the early prenatal period. Also fascinating is the fact that PFA is unique amongst the typical pediatric cerebellar tumors examined in this study in that differentiated cell types are not found, suggesting a type of differentiation block. This is perhaps furthered by the observation that PFA/PFB tumors generally respect anatomic boundaries suggesting that their ability to auto regulate themselves is partially intact. Potentially, a differentiation block could be utilized as a possible drug target as the ependymoma cells represent an early type of cell which would be absent at later stages of the development of the brain. This raises the possibility that there may be a unique check point in these tumors which could potentially be activated to arrest growth which would be absent in the more differentiated cells of the mature brain. This area requires further study as the biology of early human developmental cells is not well understood.
10. Stalled developmental programs at the root of pediatric brain tumors
Jessa, et al, Nature Genetics, December 2019, vol. 51, 1702-13
This article demonstrates that specific neural progenitor cells are a common mechanism for pediatric brain tumors. The author focused on high grade gliomas and atypical teratoid/rhabdoid tumors. A main finding of the article was that removal of an oncogenic H3k27M mutation directly promoted the progression of differentiation of tumor cells along the predicted glial lineage. This occurred despite many associated mutations in TP53 and MYC amplification. The authors argued that H3K27M had a direct effect on the differentiation potential of pontine neural progenitors. The authors argued that typical strategies utilized to target brain tumors that depend on proliferation rates and migration potential are less relevant to these tumors. The authors felt that the possibility of dedifferentiation of a more mature cell type was possible but unlikely.
Commentary: This tumor again focuses a lot of attention on the H3k27M mutation and links it with the idea that H3k27M impacts the differentiation potential of the cells in this tumor. This leads one to the conclusion that some pediatric brain tumors are unusual in that they really stalled early developmental cells that likely have a lot of their cellular machinery intact but have failed to continue along their elected developmental lineage. This is an obvious area for a drug target as you have a population of immature tumor cells which will be coexisting with cells which have developed normally.
References:
1Griesinger, et al, Neuro-Oncology 19(10), 1350-60, 2017
2J Immunother Cancer. 2018; 6: 30.
3Neuropathol Appl Neurobiol. 2001 Feb;27(1):29-39.
4Kieran, et al, Phase I study of gene-mediated cytotoxic immunotherapy with Adv-tk as adjuvant to surgery and radiation for pediatric malignant glioma and recurrent ependymoma, Neuro Oncology, 21(4), 537-546, 2019
5Pajtler, et al, Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas
6Hubner, et al, EZHIP / CXorf67 mimics K27M mutated oncohistones and functions as an intrinsic inhibitor of PRC2 function in aggressive posterior fossa ependymoma. Neuro Oncol. 2019 Mar 29.
7Pavon, et al, New Therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X, Sci Rep., 2019; 9 :9973
8Dobson et al, Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma, J Immunol 2009; 183:7428-7440
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