Highlights
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Medulloblastoma and pediatric high grade glioma cell viability is inversely correlated with HSV G207 cellular receptor expression.
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Of the thousands of differentially expressed genes after treatment with PVSIPRO or G207, only five genes were shared by both: GCLM, LANCL2, and RBM3 were enriched while ADAMTS1 and VEGFA were depleted.
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There were few differentially expressed genes between medulloblastoma and pediatric high grade glioma treated by G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1.
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Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.
Keywords: PVSRIPO, G207, Oncolytic virus, Viral immunotherapy, Medulloblastoma, Pediatric high-grade glioma, Cell response, RNA sequencing
Abstract
HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1. Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.
Introduction
Two of the most common malignant brain tumors in children are medulloblastoma and pediatric high-grade glioma (pHGG). Currently, treatment of these tumor consists of maximal safe resection typically followed by radiation and chemotherapy. However, this treatment strategy results in five-year overall survival (OS) ranging from 85% for average-risk disease to ∼50% for high-risk disease [1,2] for medulloblastoma and a median overall survival of only 16 months in pHGG [3]. The prognosis for patients with recurrent medulloblastoma and pHGG is far worse.
Viral immunotherapy is a promising alternative to conventional radiation and chemotherapy to treat recurrent medulloblastoma and pHGG. Two viral immunotherapies currently in early-phase clinical trials for the treatment of recurrent medulloblastoma and pHGG include the recombinant polio:rhinovirus chimera, PVSRIPO (NCT03043391) [4] and genetically engineered herpes simplex virus (HSV), G207 (NCT02457845 and NCT03911388) [4], [5], [6]. Additionally, PVSRIPO has been explored in adults with recurrent glioblastoma in Phase I (NCT01491893) [5] and Phase II trials (NCT02986178), while G207 has been explored in three Phase I trials alone and in combination with radiation therapy (NCT00157703) [7], [8], [9]. These studies demonstrated encouraging OS for some patients [5], however, responses have been variable, and it is unknown why some patients have excellent responses to viral immunotherapy while others do not. The molecular mechanisms governing antitumor effects of these viral immunotherapies are unclear.
We sought to explore the molecular pathways and genes elicited by PVSRIPO and G207 infection of glioma and medulloblastoma models. Using cell viability assays, flow cytometry, and RNA sequencing, we found that the two viral immunotherapies had few molecular similarities and many dissimilarities in terms of cell death, cellular viral receptor number, and cellular response to viral infection. These results imply non-overlapping mechanisms of action and sensitivities between these clinically relevant viral immunotherapy strategies.
Materials and methods
Human derived medulloblastoma and high-grade glioma cells
The medulloblastoma cell lines D324 (DAOY), D341, and D556 were obtained from the lab of Darell Bigner at Duke University. D283 were obtained from the American Type Culture Collection (ATCC). 8A-Med was obtained from the lab of Marc Remke. The pHGG line D456 was obtained from the lab of Darell Bigner (Duke University), the glioblastoma cell line U87 was obtained from the lab of Michael Brown (Duke University) and the pHGG line X21584 was obtained from the lab of Gregory Friedman. All cells were cultured in DMEM-F12 + 10%FBS, 1% NEAA, and 1% glutamine except X21584 which was cultures in neurobasal medium plus FGF, EGF, B-27 supplement, l-glutamine, amphotericin B, and gentamicin, and D341 which was cultured in Improved MEM Zinc option.
All cell lines have been previously validated in the literature. The medulloblastoma cell lines DAOY [10], D283 [11,12], D341, D556, and 8A-Med have been extensively characterized [13], [14], [15]. DAOY may be in the SHH subgroup whereas the rest are likely Group 3 subgroup [15,16]. D456 was derived from a frontal lobe glioblastoma in an 8 year old female [17]. U87 is likely a glioblastoma from an unknown origin [18,19] while X21584 was derived from a high grade glioma of a 12 year male [20].
Viral infection of tumor cells
PVSRIPO and G207 were derived and propagated as previously described [20], [21], [22]. Briefly, PVSRIPO was grown in HeLa cells [23], purified using a 0.45 µm syringe filter, concentrated and filtered again through a 100 kDa filter (Millipore). G207 was grown in Vero cells and purified on OptiPrep™ gradients (AXIS-SHIELD Oslo, Norway) [24]. Titers were determined by plaque assay [23]. The effect of PVSRIPO and G207 medulloblastoma and glioblastoma cytotoxicity was determined using CellTiter-Glo, (Promega) per the manufacturer's protocol in triplicate. Briefly, 25,000 cells in 100 µL were plated (Greiner CELLSTAR®) 96 well plates, incubated for 8 h, then treated with either PVSRIPO or G207 at multiplicity of infection (MOI) 0, 0.01, 0.1, 1, 10, or 100 for 24 h (PVSRIPO) or 72 h (G207). Cell viability line graphs were created using GraphGad Prism 9 (La Jolla, CA).
RNA sequencing of medulloblastoma and pHGG cell lines
RNA sequencing was completed as previously described [25] for D283, D324, D341, and D556 medulloblastoma and D456 and X21584 pHGG for the following G207 treatment conditions: control (0 h), 8 h after viral infection, 24 h after viral infection. RNA sequencing was completed for D283, D324, D341, and D556 medulloblastoma for the following PVSRIPO treatment conditions: control (0 h), 8 h after viral infection, 24 h after viral infection. RNA sequencing for D456 and X21584 treated with PVSRIPO was not completed due to difficulties with cell viability/processing for sequencing. The Sequencing and Genomics Technologies (SGT) Core Facility at Duke University performed RNA-sequencing. Total RNA was extracted from cell pellets using the RNeasy Plus Mini Kit (Qiagen # 74,134). mRNA was enriched from total RNA and reversed transcribed into cDNA. Kapa mRNA HyperPrep Kit (Roche # KK8581) was used to build sequencing libraries. Libraries were pooled to equal number of moles and sequenced on the NovaSeq 6000 S2 flow cell to produce 50 base pair paired-end reads. Triplicates were run for all cell types and treatment conditions, each in two lanes.
Sample fastq files quality metrics were assessed via FastQC v0.11.9 and MultiQC v1.11. Next, the paired-end reads were aligned to the GRCh38.p14 human reference genome via STAR v2.7.2b with default parameter settings. Subsequently, post-alignment quality metrics were assessed via the log file output of STAR. Quantification and generation of the raw counts matrix was performed via featureCounts v1.6.3. Replicates were combined using the collapseReplicates function within DESeq2 R package v1.36.0. The raw counts matrix was then normalized and differential expression was calculated via DeSeq2 R package v1.36.0 with a design that combined the cell line and treatment metadata. The differentially expressed genes for each comparison were then ranked by log2FC and input into fgsea R package v1.23.4 for gene set enrichment analysis along with the Hallmark gene set collection from MSigDB. RStudio 2022.07.0 Build 548 running R version 4.2.1 was used for the analysis.
The R script is located in Supplementary Materials.
Unsupervised hierarchical clustering/heatmap using the geneset GOBP_DEFENSE_RESPONSE_TO_VIRUS (https://www.gsea-msigdb.org/gsea/msigdb/cards/GOBP_DEFENSE_RESPONSE_TO_VIRUS) was created using MORPHEUS (https://software.broadinstitute.org/morpheus/).
GraphPad Prism 9 was used to calculate Pearson r of virus LD50 and receptor number and paired t-test (two tailed) of resistant vs. sensitive number of genes. Volcano plots were created using VocaNoseR [26] in which Manhattan distance was used for ranking hits.
RNA sequencing and analysis of human specimens
For patient glioblastoma specimens, RNA-seq data of samples were aligned to GRCh38 using STAR (v2.4.0.1) and expression quantification per gene was computed by FeatureCounts (v1.4.6). The function “cpm” in R package EdgeR (v3.30.3) was used to normalize the raw counts into the counts per million (CPM). The batch effects were also removed during the normalization. Specimens were obtained from the clinical trial (NCT02986178), a Phase II trial of PVSRIPO for recurrent glioblastoma in adult patients. In clinical trials that treated pediatric patients with HGG with either PVSRIPO (NCT03043391) [4] or G207 (NCT02457845) [6], there was insufficient tissue to perform RNA sequencing.
Flow cytometry
To investigate expression of CD111 and CD155, neurospheres were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies) or adherent cells were detached using Trypsin and prepared for fluorescence activated cell sorting (FACS) analysis. The following fluorochrome-conjugated monoclonal antibodies were used: PE anti-human CD111 (Nectin-1; BioLegend Cat# 340,404, RRID:AB_2,174,152) and APC anti-human CD155 (PVR; BioLegend Cat# 337,618, RRID:AB_2,565,815). Cells were analyzed with an Attune NxT Flow Cytometer (Thermo Fisher Scientific) in the UAB Flow Cytometry Core Facility, and the results were expressed as a percentage of gated cells for antibody binding using FlowJo software V10 (RRID:SCR_008520). Mean values from multiple determinations on separate dates and with separate cultures were calculated.
Results
pHGG and medulloblastoma demonstrate different sensitivities to G207 or PVSRIPO
To identify putative cellular mechanisms of resistance, we first sought to categorize medulloblastoma and pHGG cell lines as “sensitive” and “resistant” based on LD50 values after treatment with virotherapy and nonhierarchical clustering of RNA signatures. Cell viability assays were performed at standard time points (24 h for PVSRIPO and 72 h for G207) based on differences in viral replication rates. With PVSRIPO, U87, X21584, D456, D341 and D556 had less than 50% viability at an MOI 1 (Fig. 1A). Based upon these results, D341 and D556 were categorized as “sensitive” to PVSRIPO while D283 and D324 were categorized as “resistant”. These cells were thus selected to undergo RNA sequencing as described in Methods. With G207, there was a lack of a clear, natural separation of cell viability at MOI 1 (Fig. 1B); and therefore, this categorization scheme was not used for G207. D456 and X21584 were also selected for RNA sequencing as they are both pHGG cell lines.
Fig. 1.
Dose response curves of pHGG and medulloblastoma cell lines to viral immunotherapy. (A) Cell viability after treatment with PVSRIPO for 24 h. Note the separation of cell line viability at MOI 1; the cell lines D324, 8A, D425, D283 grouped together as more “resistant”. (B) Cell viability after treatment of cells with G207 for 72 h. There was no clear grouping (“resistant” vs “sensitive”) viability at MOI 1. (C) PCA plot of mRNA sequencing data of untreated medulloblastoma and pHGG cells demonstrates clustering of pHGG cells (D456, X21584). D324 or DAOY cells were distinct from other pHGG and medulloblastoma cells. (D) Unsupervised hierarchical clustering of D283, D324, D341, D556, D456, and X21584 cells using the geneset GOBP_DEFENSE_RESPONSE_TO_VIRUS, demonstrates grouping of D556/D341 cells and D283/D324 cells, similar to the grouping of the dose response curve at MOI 1 (A). MOI: multiplicity of infection.
Next, Principal Component Analysis (PCA) plot using RNA sequencing data of untreated cell types demonstrated relative clustering of the medulloblastoma cell lines D283, D341, and D556 while D324 and the pHGG cell lines D456 and X21584 had substantial variance (Fig. 1C). Nonhierarchical clustering of RNA sequencing using the geneset, GOBP_DEFENSE_RESPONSE_TO_VIRUS (http://www.gsea-msigdb.org/gsea/msigdb/cards/GOBP_DEFENSE_RESPONSE_TO_VIRUS), demonstrated adjacent clustering of cells in the following order: D556, D341, D283, D324, D456, X21584 (Fig. 1D).
Viral cellular entry molecule number correlates with cytotoxicity for G207 but not PVSRIPO
Previous work by our group demonstrated a significant inverse correlation between G207 sensitivity and expression of its cell surface receptor, nectin-1 (CD111) [20]. Flow cytometry was performed to quantify the expression of CD111 and CD155, the cell surface receptor for PVSRIPO [22]. Similar to previous findings, there was a strong inverse correlation between CD111 expression and LD50 to G207 (r = −0.985, P < 0.001, Fig. 2A). However, there was not a correlation between CD155 expression and LD50 to PVSRIPO (r = 0.111, p = 0.813, Fig. 2B) due to the near universal expression of CD155, and the exceedingly low number of CD155 moieties sufficient for virus entry [27], suggesting a different mechanism than receptor expression for decreased cytotoxicity of some cell lines to PVSRIPO. The flow cytometry gating strategy is located in Fig. S1.
Fig. 2.
Cell surface receptor expression is correlated with cytotoxicity for CD111 but not CD155. (A) Dot plot of Nectin-1 (CD111) receptor expression and Poliovirus receptor (CD155) expression by cell type as percentage of cells expressing receptor. (B) Scatter plot of flow cytometry data demonstrating Lack CD111 expression in 8A cells with robust CD155 expression vs. D556 cells demonstrating both robust CD111 and CD155 expression. (C) There is a significant inverse correlation between CD111 expression and LD50 from G207 treatment not seen in CD155 expression and LD50 from PVSRIPO treatment (D).
pHGG and medulloblastoma response to virotherapy demonstrates enrichment and depletion in multiple cellular pathways and differentially expressed genes
Medulloblastoma and pHGG cells were not treated (controls) or treated for 8 h or 24 h with either G207 or PVSRIPO. Details of all significantly enriched and depleted hallmark pathways can be found in Tables S1 and S2, respectively. Graphical representation of the most significant hallmark pathways is located in Fig. 3. Notably, the hallmark pathways with the greatest number of enrichment, E2F targets, G2M checkpoints, mitotic spindle, and myc targets all fall into the hallmark process category “proliferation” with the first three representing cell cycle progression genesets [28].
Fig. 3.
The most common enriched and depleted hallmark pathways. Bar graphs of hallmark gene sets with at least eight significantly (P ≤ 0.05) depleted (black) or enriched (gray) treatment comparisons. Treatment comparisons are on the y axis, NES on the x axis. NES: normalized enrichment score.
Medulloblastoma response to treatment with G207 and PVSRIPO differ
Details of significantly differentially expressed genes in all treatment groups can be found in Table S3. Despite the hundreds to thousands of differentially expressed genes for each treatment condition, there were only five DE genes shared by all medulloblastoma cell lines and G207 and PVSRIPO: GCLM, LANCL2, and RBM3 were enriched while ADAMTS1 and VEGFA were depleted (Table 1). Treatment with PVSRIPO was associated with a larger number of DE genes compared to G207; control vs 8 h: 187 and 8, respectively; control vs 24 h: 146 and 26, respectively (Fig. 4A). PCA plots (Fig. 4B–D) visually demonstrate the clustering of cells treated with G207 vs. PVSRIPO while a volcano plot demonstrates the interaction effects between both time points (0, 8 h, 24 h) and treatment (G207 and PVSRIPO), controlling for both (Fig. 4E).
Table 1.
Significantly Differentially Expressed Genes Enriched by G207 and PVSRIPO in Medulloblastoma.
| Enriched | ||||
| Control vs. 8 hrs | Control vs. 24 hrs | 8 hrs vs. 24 hrs | ||
| None | None | GCLM | ||
| LANCL2 | ||||
| RBM3 | ||||
| Depleted | ||||
| ADAMTS1 | VEGFA | None | ||
| Gene | Full name | Function | Reference | |
| GCLM | glutamate-cysteine ligase modifier subunit | first rate limiting enzyme of glutathione synthesis | https://www.ncbi.nlm.nih.gov/gene/2730 | |
| LANCL2 | LanC like glutathione S-transferase 2 | involved in negative regulation of transcription | https://www.ncbi.nlm.nih.gov/gene/55915 | |
| RBM3 | RNA binding motif protein 3 | member of the glycine-rich RNA-binding protein family | https://www.ncbi.nlm.nih.gov/gene/5935 | |
| ADAMTS1 | ADAM metallopeptidase with thrombospondin type 1 motif 1 | Associated with inflammation | https://www.ncbi.nlm.nih.gov/gene/9510 | |
| VEGFA | vascular endothelial growth factor A | induces proliferation and migration of vascular endothelial cells, angiogenesis | https://www.ncbi.nlm.nih.gov/gene/7422 | |
Fig. 4.
Medulloblastoma cells response differently to treatment with G207 and PVSRIPO. (A) Venn diagram demonstrating minimal overlap in G207 and PVSRIPO of significantly differentially expressed genes. PCA plots of medulloblastoma cells treated with G207 (B), PVSRIPO (C), and the two graphs combined (D). (E) Volcano plot of differentially expressed genes of cells treated with PVSRIPO vs. G207 demonstrating the interaction effect between time point and treatment, controlling for both.
Medulloblastoma and pHGG cellular response to G207 infection
Multiple significantly enriched and depleted DE genes were shared by all medulloblastoma cell lines and pHGG cell lines (Table 2). Notably, both GCLM and LANCL2 were enriched at 8- vs. 24-hour time points, two genes enriched after treatment with both PVSRIPO and G207 in medulloblastoma. Data for pHGG cellular response to PVSRIPO is not available due to cell viability/RNA processing difficulties.
Table 2.
Significantly differentially expressed genes in both medulloblastoma and HGG cell lines treated with G207.
| Gene | Gene function | Reference | |
|---|---|---|---|
| Enriched | |||
| Control vs. 8 hrs | |||
| GPSM2 | cell surface G protein modulator | https://www.ncbi.nlm.nih.gov/gene/29899 | |
| CHECK2 | checkpoint regulator of cell cycle, possible tumor suppressor | https://www.ncbi.nlm.nih.gov/gene/11200 | |
| Control vs. 24 hrs | |||
| SEPTIN2 | involved in identical protein binding including cilium, exocytosis, smoothened signaling | https://www.ncbi.nlm.nih.gov/gene/4735 | |
| 8 hrs vs. 24 hrs | |||
| EIF4G2 | involved in translation initiation | https://www.ncbi.nlm.nih.gov/gene/1982 | |
| GCLM | first rate limiting enzyme of glutathione synthesis | https://www.ncbi.nlm.nih.gov/gene/2730 | |
| GDAP1 | member of ganglioside-induced differentiation family | https://www.ncbi.nlm.nih.gov/gene/54332 | |
| LANCL2 | involved in negative regulation of transcription | https://www.ncbi.nlm.nih.gov/gene/55915 | |
| PWP1 | largely unknown, cell cycle dependent | https://www.ncbi.nlm.nih.gov/gene/11137 | |
| Depleted | |||
| Control vs. 8 hrs | |||
| LANCL2 | involved in negative regulation of transcription | https://www.ncbi.nlm.nih.gov/gene/55915 | |
| RASD1 | member of Ras family of GTPases, induced by dexamethasone | https://www.ncbi.nlm.nih.gov/gene/51655 | |
| RHOB | GTPase activity, involved in cell response to stress | https://www.ncbi.nlm.nih.gov/gene/388 | |
| Control vs. 24 hrs | |||
| ARC | involved in mRNA binding, cell migration, cytoskeleton organization | https://www.ncbi.nlm.nih.gov/gene/23237 | |
| BCL2L11 | BCL-2 family member, involved in apoptosis | https://www.ncbi.nlm.nih.gov/gene/10018 | |
| CA2 | isozyme of carbonic anhydrase | https://www.ncbi.nlm.nih.gov/gene/760 | |
| CBX4 | enables small ubiquitin-like modifier (SUMO) binding activity | https://www.ncbi.nlm.nih.gov/gene/8535 | |
| CCDC137 | enables RNA binding | https://www.ncbi.nlm.nih.gov/gene/339230 | |
| CCN1 | promotes cell adhesion | https://www.ncbi.nlm.nih.gov/gene/3491 | |
| CCN2 | involved in cell adhesion | https://www.ncbi.nlm.nih.gov/gene/1490 | |
| EGR2 | transcription factor | https://www.ncbi.nlm.nih.gov/gene/1959 | |
| FRG2C | unknown, located in nucleus | https://www.ncbi.nlm.nih.gov/gene/100288801 | |
| GADD45B | respond to cellular stress | https://www.ncbi.nlm.nih.gov/gene/4616 | |
| JUNB | involved in transcription by RNA polymerase II | https://www.ncbi.nlm.nih.gov/gene/3726 | |
| LOC124900276 | "cuticle collagen 2-like", unknown | https://www.ncbi.nlm.nih.gov/gene/?term=LOC124900276 | |
| MYLIP | interacts with myosin regulatory light chain | https://www.ncbi.nlm.nih.gov/gene/29116 | |
| NFKBIA | member of NF-kappa-B inhibitor family | https://www.ncbi.nlm.nih.gov/gene/4792 | |
| NXF1 | member of nuclear RNA export factors | https://www.ncbi.nlm.nih.gov/gene/10482 | |
| RASL11B | member of GTPase family similar to RAS | https://www.ncbi.nlm.nih.gov/gene/65997 | |
| RGS2 | GTPase activating protein | https://www.ncbi.nlm.nih.gov/gene/5997 | |
| SGK1 | kinase with role cellular stress response | https://www.ncbi.nlm.nih.gov/gene/6446 | |
| ZNF296 | enables DNA binding activity | https://www.ncbi.nlm.nih.gov/gene/162979 | |
| 8 hrs vs. 24 hrs | |||
| CBX4 | see above | ||
| CCN1 | see above | ||
| JUNB | see above | ||
| LSMEM1 | unknown, possible membrane component | https://www.ncbi.nlm.nih.gov/gene/286006 | |
| MUC3A | epithelial glycoprotein, described in gastrointestinal cancer | https://www.ncbi.nlm.nih.gov/gene/4584#gene-expression | |
| NRARP | involved in Notch and Wnt signaling pathways | https://www.ncbi.nlm.nih.gov/gene/441478 | |
| NXF1 | see above | ||
| RASD1 | see above | ||
| RSRP1 | implicated in glioblastoma mesenchymal phenotype | PMID: 34,042,961 | |
| ZNF296 | see above |
Resistant vs. sensitive cellular response to PVSRIPO
Comparing untreated “resistant (D283, D324)” and “sensitive (D341, D556)” medulloblastoma cells, there were over 4000 significantly differentially expressed genes. The top 50 are highlighted in a volcano plot (Fig. 5A), demonstrating many more enriched signatures in the “sensitive cells”. Treatment of “resistant” compared to “sensitive” medulloblastoma cell lines with PVSRIPO demonstrated a significantly lower number of resistant cell line depleted genes (Fig. 5B). Likewise, there was a decreased number of resistant cell line enriched genes (Fig. 5C), however, this difference did not achieve statistical significance.
Fig. 5.
Genetic activation is preferentially increased in “sensitive” medulloblastoma cell lines. (A) Volcano plot of untreated “resistant” medulloblastoma cells (D283 and D324) vs. “sensitive” (D341 and D556) cells demonstrates many more significantly more enriched differentially expressed cells in the sensitive group. (B) Line graphs demonstrating all significantly depleted genes in sensitive vs. resistance medulloblastoma cells. There were significantly more gene depletions in sensitive cells. (C) Line graphs demonstrating all significantly enriched genes in sensitive vs. resistant medulloblastoma cells with a trend toward more genes in the sensitive group.
Shared genes in poor survival in a clinical trial of glioblastoma and “resistant” medulloblastoma cell lines
We first analyzed RNA sequencing of patient recurrent glioblastoma samples taken prior to treatment with PVSRIPO in a Phase II clinical trial (“PVSRIPO in Recurrent Malignant Glioma”, NCT02986178, https://www.clinicaltrials.gov/study/NCT02986178?term=pvsripo&page=1&rank=1). Patients were categorized by < or ≥ one year OS after treatment with PVSRIPO. “Low” survival was defined as < one year OS. The top 50 differentially expressed genes can be found in Table S4, Fig. 6. The only significantly different geneset in the comparison of these groups was the HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION [29,30], adjusted P<0.001, NES 1.5949.
Fig. 6.
Volcano plot of top 50 differentially expressed genes in < and ≥ 1 year survival glioblastoma patient samples. Genes in blue are depleted in patients living < 1 year (N = 28). Genes in red are enriched in patients living ≥ 1 year (N = 29).
Next, we analyzed DE genes shared by both PVSRIPO “resistant” medulloblastoma and “low” survival in a Phase II trial of PVSRIPO for recurrent glioblastoma (NCT02986178) to determine the presence of any shared genes from each dataset. The following genes were enriched in both datasets: CCDC13, LGR6, LMO2, LUM, and RARB while the following genes were depleted in both datasets: ADAMTS20, ANGPTL4, BEND, CSF1, FUT11, PCSK6, PLIN2, and PLS1 (Table 3). Notably, none of these genes were found in the “low” vs. “high” survival of the Phase II study indicating another putative set of biomarkers to predict response to PVSRIPO across tumor types.
Table 3.
Differentially expressed genes shared by "low survival" glioblastoma and "resistant" medulloblastoma.
| Genes: enriched | Full name | Function | Reference |
|---|---|---|---|
| CCDC13 | coiled-coil domain containing 13 | DNA damage response; microtubule and cilium organization | https://www.ncbi.nlm.nih.gov/gene/152206 |
| LGR6 | leucine rich repeat containing G protein-coupled receptor 6 | G coupled protein hormone receptor | |
| LMO2 | LIM domain only 2 | Role in hematopoietic development | https://www.ncbi.nlm.nih.gov/gene/4005 |
| LUM | lumican | Member of small leucine-rich proteoglycan family, regulates spacings in collagen | https://www.ncbi.nlm.nih.gov/gene/4060 |
| RARB | retinoic acid receptor beta | Mediates embryonic morphogenesis, cell differentiation/growth | https://www.ncbi.nlm.nih.gov/gene/5915 |
| Genes: depleted | |||
| ADAMTS20 | ADAM metallopeptidase with thrombospondin type 1 motif 20 | Possibly involved in tissue remodeling | https://www.ncbi.nlm.nih.gov/gene/80070 |
| ANGPTL4 | angiopoietin like 4 | Serum hormone regulating insulin sensitivity, lipid metabolism, glucose homeostasis; endothelial cell apoptosis survival factor | https://www.ncbi.nlm.nih.gov/gene/51129 |
| BEND4 | BEN domain containing 4 | Possibly involved in DNA binding | https://www.ncbi.nlm.nih.gov/gene/389206 |
| CSF1 | colony stimulating factor 1 | Cytokine involved in macrophage function and differentiation | https://www.ncbi.nlm.nih.gov/gene/1435 |
| FUT11 | fucosyltransferase 11 | Possibly involved in fucosylation | https://www.ncbi.nlm.nih.gov/gene/170384 |
| PCSK6 | proprotein convertase subtilisin/kexin type 6 | Processes peptide precursors in the secretory pathway | https://www.ncbi.nlm.nih.gov/gene/5046 |
| PLIN2 | perilipin 2 | Coat lipid storage droplets intracellularly | https://www.ncbi.nlm.nih.gov/gene/123 |
| PLS1 | plastin 1 | Actin binding proteins in most tissues | https://www.ncbi.nlm.nih.gov/gene/5357 |
Discussion
Both viral recombinants investigated in this study have the capacity to infect cancerous cells derived of malignant gliomas or medulloblastomas, by virtue of expression of polio- and HSV-1 entry receptors on such cellular hosts. However, the relative contributions of viral targeting of the neoplastic compartment to antitumor immunity likely differ considerably for both agents.
PVSRIPO primarily targets myeloid antigen presenting cells (dendritic cells/ macrophages) in patient ex vivo glioma slices [31] and in mouse glioma models [31]. Infection of the tumor myeloid infiltrate/CNS resident myeloid compartments—producing profound immune stimulation through endogenous, sustained type-I interferon release—is sufficient for generating tumor immune surveillance in mouse tumor models [21,[31], [32], [33]]. In contrast, immunotherapy mechanisms of oncolytic HSV-1 recombinants were shown to rely on cytopathogenic infection of neoplastic cells with a potential secondary role for myeloid bystanders [34].
In this work we analyzed the outcome of infection with either G207 or PVSRIPO in a panel of pHGG and medulloblastoma cell lines. We found a significant correlation between CD111 and lethality of cells treated with G207. This confirms our previous results in which a host of adult and pediatric HGG as well as embryonal tumors treated with G207 demonstrated a significant inverse correlation between CD111 and LD50 [20]. However, we did not find any correlation between CD155 expression and PVSRIPO lethality due to the universal high percentage of cells expressing CD155. Future research will determine if CD111 in tumor specimen will predict clinical outcomes in children with HGG and medulloblastoma.
A main objective of this study was to determine if there are common responses in infected cancerous cells to disparate viral immunotherapies, HSV G207, a double stranded DNA virus and the polio:rhinovirus recombinant, PVSRIPO, a single positive-strand RNA virus. Of the 32,117 genes analyzed, only five were found to be significantly shared by both: GCLM, LANCL2, and RBM3 were enriched while ADAMTS1 and VEGFA were depleted. Table 1 contains a brief description of their function, however, there are no obvious commonalities amongst these genes. Notably, there is an increasing body of literature describing inactions of VEGF and viral immunotherapy; combining anti-VEGF treatment with virus increases outcomes in mice models compared to either alone [35], [36], [37]. Notably, GCLM, LANCL2 were also significantly shared between pHGG and medulloblastoma cell treated with G207. Glutamate-cysteine ligase modifier subunit (GCLM) is the first rate limiting enzyme in glutathione synthesis while LanC-like protein 2 (LANCL2) is a negative transcription regulator. To our knowledge, there are no therapeutics that specifically target GCLM or LANCL2 thereby making the assessment of these entities challenging to assess if modulation can result in improved viral replication and lysis throughout a tumor.
Using data from a Phase II trial of PVSRIPO for recurrent adult glioblastoma, we highlighted genes that were both enriched or depleted in patients surviving greater than one year compared to less than 1 year. Notably, enrichment of the HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION was associated with poor survival. This is somewhat expected given the 200 genes are associated with the epithelial mesenchymal transition in cancer, suggestive of more aggressive tumors that are likely less responsive to viral immunotherapy [30]. Additionally, we found the following genes in both PVSRIPO “resistant” medulloblastoma and “low” survival after treatment with PVSRIPO: CCDC13, LGR6, LMO2, LUM, RARB, ADAMTS20, ANGPTL4, BEND, CSF1, FUT11, PCSK6, PLIN2, and PLS1. It is noteworthy that colony stimulating factor 1 (CSF1), a cytokine controlling macrophage function, was depleted in these cohorts. Our group has found that higher myeloid cell density was associated with lower tumor mutational burden which was associated with longer survival after PVSRIPO administration [38]. Further work will determine if low CSF1 could be a clinical biomarker predicting poor response to viral immunotherapy.
In the context of Phase I clinical trials exploring PVSRIPO and G207 to treat malignant pediatric brain tumors, it is noteworthy that in both trials, only patients with recurrent pediatric high grade gliomas were enrolled [4,6]. Therefore, it is unknown if clinical responses and overall survival would differ in patients with newly diagnosed pHGG. In recurrent HGG, T cells are enriched in the perivascular region and immunosuppressive regulatory T cells are decreased [39]. Additionally, recurrent HGG have more tumor-infiltrating lymphocytes, macrophages, and PD-1 positive cells [40]. However, patients have profoundly decreased lymphocyte counts after chemoradiation. In a manuscript which analyzed data from four different clinical studies, lymphocyte counts were normal in 83% of patient before treatment compared to only 43% after chemoradiotherapy [41]. While some tumor characteristics may enhance immunotherapy at recurrence, profound systemic lymphopenia may severely limit the ability for the systemic immune system to respond. Additional studies of G207 and PVSRIPO to treat patients in the newly diagnosed setting are needed to resolve this uncertainty.
Limitations to our findings are as follow: 1) experiments using cell lines may have limited translational applicability, 2) “low” survival does not account for the multitude of other possible predictors of outcomes in glioblastoma clinical trials, and 3) the intrinsic limitations analyzing medulloblastoma cell line data with clinical data from an adult glioblastoma clinical trial. However, the finding of these overlapping genes in different tumor types in both in vitro and clinic setting is also a strength that may indicate these genes are putative mediators of resistance to PVSRIPO. Furthermore, although the Phase 1b clinical trial of PVSRIPO for pediatric malignant brain tumors (NCT03043391) was open to patients with medulloblastoma as well as pHGG, only patients with pHGG were enrolled [4]. Therefore, no clinical data analyzing medulloblastoma cellular response to PVSRIPO was available to compare/contrast to the in vitro data in this work. Notably, the ongoing trial of G207 for pediatric cerebellar tumors (NCT03911388) is enrolling patients with medulloblastoma [29] and may provide additional insight into the findings of medulloblastoma cell lines treated with G207 in this work. Together, this work provides basic foundational data as to the mechanism of viral immunotherapy in pHGG and medulloblastoma and serves as the starting point to explore identified genes in further detail in order to optimize G207 and PVSRIPO for the treatment of malignant brain tumors.
CRediT authorship contribution statement
Eric M. Thompson: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Kyung-Don Kang: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. Kevin Stevenson: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – review & editing. Hengshan Zhang: Data curation, Investigation, Methodology, Validation, Writing – review & editing. Matthias Gromeier: Resources, Writing – review & editing. David Ashley: Resources, Writing – review & editing. Michael Brown: Data curation, Methodology, Resources, Validation, Writing – review & editing. Gregory K. Friedman: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.
Declaration of competing interest
Two authors, Michael Brown and Matthias Gromeier own intellectual property related to PVSRIPO, which has been licensed to Istari Oncology, Inc. Matthias Gromeier holds equity in Istari Oncology, Inc. Drs. Brown and Gromeier received consultancy fees from Istari Oncology, Inc.
Acknowledgments
Funding: American Brain Tumor Association, The Cure Starts Now, Department of Defense (CA171067), The Musella Foundation, Chetna and Meena Trust (EMT). Rally Foundation for Childhood Cancer Research, CureSearch for Children's Cancer, The V Foundation for Cancer Research, Andrew McDonough B+ Foundation, National Pediatric Cancer Foundation, and Pediatric Cancer Research Foundation (GKF). NIH P30 AR048311 and NIH P30 AI27667 grants (UAB Comprehensive Flow Cytometry Core Facility).
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.101875.
Appendix. Supplementary materials
Data availability
RNA sequencing data from G207 and PVSRIPO treated cell lines is available at GSE233949. Other data will be considered by the authors on a case-by-case basis under a material transfer agreement.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
RNA sequencing data from G207 and PVSRIPO treated cell lines is available at GSE233949. Other data will be considered by the authors on a case-by-case basis under a material transfer agreement.