Abstract
INTRODUCTION
Comprising approximately 8% of our genome, Human Endogenous RetroViruses (HERVs) represent a class of germline retroviral infections that are regulated through epigenetic ...modifications. In cancer cells, which often have epigenetic dysregulation, HERVs have been implicated as potential oncogenic drivers. However, their role in gliomas is not known.Given the link between HERV expression in cancer cell lines and the distinct epigenetic dysregulation in gliomas, we utilized a tailored bioinformatic pipeline to characterize and validate the glioma retrotranscriptome and correlate HERV expression with locus-specific epigenetic modifications.Method:A custom workflow was used to quantify HERV expression in our cell lines of interest. Cell-line methylation was quantified using a custom script. We generated primers specific for the Human endogenous Mouse mammary tumor (MMTV)-Like virus 6 (HML-6). Visualization of RNA transcripts was performed using RNA-scope. Clinical data was obtained using the R package, TCGABiolinks.
RESULTS
The A172 cell line had significantly higher mean overall HERV expression relative to the M059J and H4 cell lines (p< 0.0001 for both). A172 cells had significantly lower mean number of CpG islands relative to M059J cells and H4 cells (p< 0.0001 for both). There was a significant inverse correlation between mean beta value and FC HERV expression (R=-0.57, p=0.01). qPCR confirmed robust expression of the HML-6 locus in cell culture and neurospheres. Elevated ERVK3-1 expression was associated reduced survival among IDHwt GBM patients (18.3 vs. 15.1 months, p=0.039). This was preserved among IDH mutant (IDHm) GBM as well (17.9 months vs. 14.0 months, p=0.0088).
CONCLUSION
In gliomas, HERV expression correlates with loss of DNA methylation at HERV loci. HML-6 is overexpressed in highly invasive glioblastoma cell lines and patient-derived neurospheres. We have demonstrated a potential survival detriment associated with elevated expression of the HML-6 product, ERVK3-1.
Abstract
Atypical teratoid rhabdoid tumor (ATRT) is a pediatric brain tumor with a high mortality rate characterized by mutations in/ deletions of SWI/SNF matrix-associated actin-dependent regulator ...of chromatin sub-family B member 1 (SMARCB1). We previously showed that loss of SMARCB1 causes up-regulation and release of HML-2 subfamily of human endogenous retrovirus K envelope protein (HML-2 ENV), resulting in maintenance of pluripotency. Here, we investigated intracellular trafficking and release of HML-2 ENV. Further, we demonstrate two potential therapeutic strategies to decrease intracellular HML-2 ENV: 1) inhibition of calcium influx by ouabain, a cardiac glycoside toxic to neural stem cells, and 2) targeted inhibition of cyclin-dependent kinase 5 (CDK5), which is restricted to neurons by p35, its activator protein, by TP5. ATRT cell lines and tumor tissue obtained from patients were confirmed for SMARCB1 loss and increased HML-2 ENV. Cell viability and intracellular HML-2 ENV concentration were measured after treatment with ouabain and TP5 (CDK5 antagonist). We evaluated the calcium-mediated effect of ouabain on HML-2 intracellular concentration by treating the cells with ouabain, the calcium chelators calcimycin and EGTA, and calpeptin, a calpain inhibitor, which activates CDK5, and measuring HML-2 ENV and p35. We evaluated HML-2 ENV for a CDK5 consensus phosphorylation site and performed co-immunoprecipitation to evaluate direct interaction. We evaluated activity of CDK5 in ATRT cell lines by autoradiography. Both Ouabain and TP5 caused a decrease in cell viability in a dose-dependent manner. Further, ouabain treatment decreased HML-2 ENV intracellular concentration. We found that HML-2 ENV contains a consensus phosphorylation site for CDK5. We discovered that HML-2 ENV was bound to CDK5. We established that ATRT cell lines had hyperactive CDK5. Finally, we established that the effect of ouabain on HML-2 ENV was due to indirect inhibition of calcium-mediated activation of calpain and thus CDK5.
Amyotrophic lateral sclerosis (ALS) is a universally fatal neurodegenerative disease with no cure. Human endogenous retroviruses (HERVs) have been implicated in its pathogenesis but their relevance ...to ALS is not fully understood. We examined bulk RNA-seq data from almost 2,000 ALS and unaffected control samples derived from the cortex and spinal cord. Using different methods of feature selection, including differential expression analysis and machine learning, we discovered that transcription of HERV-K loci 1q22 and 8p23.1 were significantly upregulated in the spinal cord of individuals with ALS. Additionally, we identified a subset of ALS patients with upregulated HERV-K expression in the cortex and spinal cord. We also found the expression of HERV-K loci 19q11 and 8p23.1 was correlated with protein coding genes previously implicated in ALS and dysregulated in ALS patients in this study. These results clarify the association of HERV-K and ALS and highlight specific genes in the pathobiology of late-stage ALS.
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•HML-2 loci 1q22 and 8p23.1 were upregulated in the spinal cord of ALS patients•A high HERV-K expressing ALS subgroup (20% of patients) was identified•8p23.1 expression is associated with the expression of ALS-associated genes•CA1, CXCL11, and ERVL-E could be important biomarkers of ALS
Biocomputational method; Bioinformatics; Biological sciences; Health informatics; Medical informatics; Natural sciences
INTRODUCTION:
Human Endogenous Retrovirus (HERV) are ancestral viral relics that comprise nearly 8% of the human genome. Although silenced in normal tissues, the most recently integrated provirus ...HERV-K (HML-2) can be pathologically reactivated in certain cancers.
METHODS:
We utilized a combination approach using scRNA-seq, multiplex immunofluorescence, and TEM to characterize HERV-K expression in malignant gliomas. Using CRISPR engineering and CHIP-qPCR, we downregulated HML-2 in both patient-derived glioma neurospheres and intracranial orthotopic models and identified interactions between HERV-K and pluirpotency transcription factors.
RESULTS:
We identified pathological expression of HML-2 in human malignant gliomas in CSF (HERV-K DNA/RPP30 = 35.2 ± 8.8 vs 23.1 ± 6.7, n = 18, p = 0.02) and tumors (HERV-K RNA/HPRT mean = 1.15 ± 0.2 vs. 0.5 ± 0.2, p = 0.01, n = 20) compared to epilepsy controls. Aberrant HML-2 expression corresponded to a unique stem-cell niche using multivoxel automated segmentation. Using a tailored scRNA-seq sequencing pipeline, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells that can drive cellular plasticity (ANOVA, p < 0.001). Using CRISPR technology, we demonstrate that HML-2 is critical to maintenance of glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models (OS: 26 days vs. 18.6, p=0.0008, n=20). Downregulation of HERV-K using CRISPRi reduces the glioblastoma neurosphere formation (2-way ANOVA, p < 0.0001) and HERV-K env, Polymerase, OCT4 and Nestin transcripts (2-way ANOVA p < 0.001). Moreover, using Transmission Electron Microscopy, we discovered that some glioma stem-cells form immature retroviral virions in glioblastoma.
Inhibiting HML-2 expression with nucleoside reverse transcriptase inhibitors reduces extracellular reverse transcriptase (One-way ANOVA, p < 0.05), tumor viability (IC50 = 75.8-123.1 uM), and pluripotency (1-way ANOVA, p < 0.01).
CONCLUSIONS:
Our results suggest that HML-2 is overexpressed in the glioblastoma stem-cell niche. Since persistence of glioblastoma stem-cells is considered responsible for treatment resistance/recurrence, HML-2 may serve as a unique therapeutic target in glioblastoma.
Abstract
Human Endogenous Retrovirus (HERV) are ancestral viral relics that comprise nearly 8% of the human genome. Although silenced in normal tissues, the most recently integrated provirus HERV-K ...(HML-2) can be pathologically reactivated in certain cancers. Here, we report pathological expression of HML-2 transcripts in human malignant gliomas in cerebrospinal fluid (HERV-K DNA/RPP30=35.2±8.8 vs 23.1±6.7, n=18, p=0.02) and tumors (HERV-K RNA/HPRTmean=1.15±0.2 vs. 0.5±0.2, p=0.01, n=20) compared to epilepsy controls. Aberrant HML-2 expression corresponded to a unique stem-cell niche using multivoxel automated segmentation. Using a tailored single-cell RNA sequencing pipeline to detect retrotransposons, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells that can drive cellular plasticity (ANOVA, multiple-testing correction, p< 0.001). Using CRISPR technology, we demonstrate that HML-2 is critical to maintenance of glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models (OS: 26 days vs. 18.6, p=0.0008, n=20). Downregulation of HERV-K using CRISPRi reduces the glioblastoma neurosphere formation (2-way ANOVA, p< 0.0001). and HERV-K env, Polymerase, OCT4 and Nestin transcripts (2-way ANOVA p< 0.001). Using Chromatin Immunoprecipitation, we determined that this interaction in gliomas is likely mediated by the nuclear transcription factor OCT4 which binds to an HML-2 specific Long-Terminal Repeat (LTR5Hs). Moreover, using Transmission Electron Microscopy, we discovered that some glioma stem-cells form immature retroviral virions in glioblastoma. Inhibiting HML-2 expression with nucleoside reverse transcriptase inhibitors reduces extracellular reverse transcriptase (One-way ANOVA, p< 0.05), tumor viability (IC50=75.8-123.1 uM), and pluripotency (One-way ANOVA, p< 0.01). Our results suggest that HML-2 is overexpressed in the cancer stem-cell niche of glioblastoma. Since persistence of glioblastoma stem-cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target in glioblastoma.
