Antiretroviral Drug Repositioning for Glioblastoma.

Outcomes for glioblastoma (GBM) remain poor despite standard-of-care treatments including surgical resection, radiation, and chemotherapy. Intratumoral heterogeneity contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. Drug repositioning studies on antiretroviral therapy (ART) have shown promising potent antineoplastic effects in multiple cancers; however, its efficacy in GBM remains unclear. To better understand the pleiotropic anticancer effects of ART on GBM, we conducted a comprehensive drug repurposing analysis of ART in GBM to highlight its utility in translational neuro-oncology. To uncover the anticancer role of ART in GBM, we conducted a comprehensive bioinformatic and in vitro screen of antiretrovirals against glioblastoma. Using the DepMap repository and reversal of gene expression score, we conducted an unbiased screen of 16 antiretrovirals in 40 glioma cell lines to identify promising candidates for GBM drug repositioning. We utilized patient-derived neurospheres and glioma cell lines to assess neurosphere viability, proliferation, and stemness. Our in silico screen revealed that several ART drugs including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) demonstrated marked anti-glioma activity with the capability of reversing the GBM disease signature. RTIs effectively decreased cell viability, GBM stem cell markers, and proliferation. Our study provides mechanistic and functional insight into the utility of ART repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, ARTs may be a promising adjuvant treatment for GBM.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Epigenetic Regulation in Primary CNS Tumors: An Opportunity to Bridge Old and New WHO Classifications.

Originally approved in 1979, a specific grading classification for central nervous system (CNS) tumors was devised by the World Health Organization (WHO) in an effort to guide cancer treatment and better understand prognosis. These "blue books" have since undergone several iterations based on tumor location, advancements in histopathology, and most recently, diagnostic molecular pathology in its fifth edition. As new research methods have evolved to elucidate complex molecular mechanisms of tumorigenesis, a need to update and integrate these findings into the WHO grading scheme has become apparent. Epigenetic tools represent an area of burgeoning interest that encompasses all non-Mendelian inherited genetic features affecting gene expression, including but not limited to chromatin remodeling complexes, DNA methylation, and histone regulating enzymes. The SWItch/Sucrose non-fermenting (SWI/SNF) chromatin remodeling complex is the largest mammalian family of chromatin remodeling proteins and is estimated to be altered in 20-25% of all human malignancies; however, the ways in which it contributes to tumorigenesis are not fully understood. We recently discovered that CNS tumors with SWI/SNF mutations have revealed an oncogenic role for endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline and are inherited like Mendelian genes, several of which retain open reading frames for proteins whose expression putatively contributes to tumor formation. Herein, we analyzed the latest WHO classification scheme for all CNS tumors with documented SWI/SNF mutations and/or aberrant ERV expression, and we summarize this information to highlight potential research opportunities that could be integrated into the grading scheme to better delineate diagnostic criteria and therapeutic targets.

Does the interplay between human endogenous retrovirus K and extracellular vesicles contribute to aging?

The role of extracellular vesicles (EVs), including retroviral-like particles (RVLPs), in pathogenic processes is currently a subject of active investigation. Several studies have identified mechanistic links between the increased presence of EVs and the process of senescence. A recent study reveals that the reverse transcribed complementary DNA (cDNA) of a human endogenous retroviral sequence can activate the innate immune system and result in tissue damage and/or the spread of cellular senescence to distant tissues. Several studies have linked EVs to age-related diseases, such as Alzheimer's disease and Parkinson's disease, and have included isolation of EVs from individuals with these diseases. Loss of epigenetic regulation, immune activation, and environmental stimuli can all lead to the expression of endogenous retroviruses and the incorporation of their proteins and transcripts into EVs. In addition, EVs disseminating these endogenous retroviral components have now been shown to act in a paracrine manner in multiple human diseases. Further investigation of the connection between EVs containing endogenous retroviral protein products or nucleotides should be pursued in models of age-related diseases.

The Role of HERV-K in Cancer Stemness.

Human endogenous retrovirus-K (HERV-K) is the most recently integrated retrovirus in the human genome, with implications for multiple disorders, including cancer. Although typically transcriptionally silenced in normal adult cells, dysregulation of HERV-K (HML-2) elements has been observed in cancer, including breast, germ cell tumors, pancreatic, melanoma, and brain cancer. While multiple methods of carcinogenesis have been proposed, here we discuss the role of HERV-K (HML-2) in the promotion and maintenance of the stem-cell in cancer. Aberrant expression of HERV-K has been shown to promote expression of stem cell markers and promote dedifferentiation. In this review, we discuss HERV-K (HML-2) as a potential therapeutic target based on evidence that some tumors depend on the expression of its proteins for survival.

Differential expression of an endogenous retroviral element [HERV-K(HML-6)] is associated with reduced survival in glioblastoma patients.

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. We identified robust overexpression of multiple HERVs in our cell lines, including a retroviral transcript, HML-6, at 19q13.43b in glioblastoma cells. HERV expression inversely correlated with loci-specific DNA methylation. HML-6 contains an intact open reading frame encoding a small envelope protein, ERVK3-1. Increased expression of ERVK3-1 in GBM patients is associated with a poor prognosis independent of IDH-mutational status. Our results suggest that not only is HML-6 uniquely overexpressed in highly invasive cell lines and tissue samples, but also its gene product, ERVK3-1, may be associated with reduced survival in GBM patients. These results may have implications for both the tumor biology of GBM and the role of ERVK3-1 as a potential therapeutic target.

A multiparametric pharmacogenomic strategy for drug repositioning predicts therapeutic efficacy for glioblastoma cell lines.

BACKGROUND: Poor prognosis of glioblastoma patients and the extensive heterogeneity of glioblastoma at both the molecular and cellular level necessitates developing novel individualized treatment modalities via genomics-driven approaches. METHODS: This study leverages numerous pharmacogenomic and tissue databases to examine drug repositioning for glioblastoma. RNA-seq of glioblastoma tumor samples from The Cancer Genome Atlas (TCGA, n = 117) were compared to "normal" frontal lobe samples from Genotype-Tissue Expression Portal (GTEX, n = 120) to find differentially expressed genes (DEGs). Using compound gene expression data and drug activity data from the Library of Integrated Network-Based Cellular Signatures (LINCS, n = 66,512 compounds) CCLE (71 glioma cell lines), and Chemical European Molecular Biology Laboratory (ChEMBL) platforms, we employed a summarized reversal gene expression metric (sRGES) to "reverse" the resultant disease signature for GBM and its subtypes. A multiparametric strategy was employed to stratify compounds capable of blood-brain barrier penetrance with a favorable pharmacokinetic profile (CNS-MPO). RESULTS: Significant correlations were identified between sRGES and drug efficacy in GBM cell lines in both ChEMBL(r = 0.37, P < .001) and Cancer Therapeutic Response Portal (CTRP) databases (r = 0.35, P < 0.001). Our multiparametric algorithm identified two classes of drugs with highest sRGES and CNS-MPO: HDAC inhibitors (vorinostat and entinostat) and topoisomerase inhibitors suitable for drug repurposing. CONCLUSIONS: Our studies suggest that reversal of glioblastoma disease signature correlates with drug potency for various GBM subtypes. This multiparametric approach may set the foundation for an early-phase personalized -omics clinical trial for glioblastoma by effectively identifying drugs that are capable of reversing the disease signature and have favorable pharmacokinetic and safety profiles.

Endogenous Retroviral Elements in Human Development and Central Nervous System Embryonal Tumors.

Human endogenous retroviruses (HERVs), which are critical to normal embryologic development and downregulated during normal maturation, have been implicated in a variety of cancers. Abnormal persistent production of HERVs has been suggested to play a role in oncogenesis and to confer stem cell properties to cells. We recently demonstrated that the most recently incorporated HERV element (HERV-K HML-2) has been associated with the pathogenesis of the embryonal atypical teratoid rhabdoid tumor (AT/RT), shifting our understanding of embryonal tumor development. HML-2 expression is vital for proper human development and its expression is suppressed via methylation or chromatin remodeling as cells differentiate. We previously found that dysfunctional chromatin remodeling due to loss of SMARCB1 expression induces HML-2 envelope (env) expression, impairing cellular differentiation and migration, and facilitating tumor growth in AT/RT. Epigenetic dysregulation in other embryonal tumors with concomitant expression of stem-cell markers may facilitate HML-2 expression. Future studies could utilize HML-2 as potential diagnostic criteria, use its expression as a treatment biomarker, and investigate the efficacy of therapies targeting cells with high HML-2 expression.

SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway.

Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.

Reading LINEs within the cocaine addicted brain.

INTRODUCTION: Long interspersed element (LINE)-1 (L1) is a type of retrotransposon capable of mobilizing into new genomic locations. Often studied in Mendelian diseases or cancer, L1s may also cause somatic mutation in the developing central nervous system. Recent reports showed L1 transcription was activated in brains of cocaine-treated mice, and L1 retrotransposition was increased in cocaine-treated neuronal cell cultures. We hypothesized that the predisposition to cocaine addiction may result from inherited L1s or somatic L1 mobilization in the brain. METHODS: Postmortem medial prefrontal cortex (mPFC) tissue from 30 CA and 30 control individuals was studied. An Alexafluor488-labeled NeuN antibody and fluorescence activated nuclei sorting were used to separate neuronal from non-neuronal cell nuclei. L1s and their 3' flanking sequences were amplified from neuronal and non-neuronal genomic DNA (gDNA) using L1-seq. L1 DNA libraries from the neuronal gDNA were sequenced on an Illumina HiSeq2000. Sequences aligned to the hg19 human genome build were analyzed for L1 insertions using custom "L1-seq" bioinformatics programs. RESULTS: Previously uncataloged L1 insertions, some validated by PCR, were detected in neurons from both CA and control brain samples. Steady-state L1 mRNA levels in CA and control mPFC were also assessed. Gene ontology and pathway analyses were used to assess relationships between genes putatively disrupted by novel L1s in CA and control individuals. L1 insertions in CA samples were enriched in gene ontologies and pathways previously associated with CA. CONCLUSIONS: We conclude that neurons in the mPFC harbor L1 insertions that have the potential to influence predisposition to CA.

Somatically Acquired LINE-1 Insertions in Normal Esophagus Undergo Clonal Expansion in Esophageal Squamous Cell Carcinoma.

Squamous cell carcinoma of the esophagus (SCC) is the most common form of esophageal cancer in the world and is typically diagnosed at an advanced stage when successful treatment is challenging. Understanding the mutational profile of this cancer may identify new treatment strategies. Because somatic retrotransposition has been shown in tumors of the gastrointestinal system, we focused on LINE-1 (L1) mobilization as a source of genetic instability in this cancer. We hypothesized that retrotransposition is ongoing in SCC patients. The expression of L1 encoded proteins is necessary for retrotransposition to occur; therefore, we evaluated the expression of L1 open reading frame 1 protein (ORF1p). Using immunohistochemistry, we detected ORF1p expression in all four SCC cases evaluated. Using L1-seq, we identified and validated 74 somatic insertions in eight tumors of the nine evaluated. Of these, 12 insertions appeared to be somatic, not genetically inherited, and sub-clonal (i.e., present in less than one copy per genome equivalent) in the adjacent normal esophagus (NE), while clonal in the tumor. Our results indicate that L1 retrotransposition is active in SCC of the esophagus and that insertion events are present in histologically NE that expands clonally in the subsequent tumor.

LINE-1 expression and retrotransposition in Barrett's esophagus and esophageal carcinoma.

Barrett's esophagus (BE) is a common disease in which the lining of the esophagus transitions from stratified squamous epithelium to metaplastic columnar epithelium that predisposes individuals to developing esophageal adenocarcinoma (EAC). We hypothesized that BE provides a unique environment for increased long-interspersed element 1 (LINE-1 or L1) retrotransposition. To this end, we evaluated 5 patients with benign BE, 5 patients with BE and concomitant EAC, and 10 additional patients with EAC to determine L1 activity in this progressive disease. After L1-seq, we confirmed 118 somatic insertions by PCR in 10 of 20 individuals. We observed clonal amplification of several insertions which appeared to originate in normal esophagus (NE) or BE and were later clonally expanded in BE or in EAC. Additionally, we observed evidence of clonality within the EAC cases; specifically, 22 of 25 EAC-only insertions were present identically in distinct regions available from the same tumor, suggesting that these insertions occurred in the founding tumor cell of these lesions. L1 proteins must be expressed for retrotransposition to occur; therefore, we evaluated the expression of open reading frame 1 protein (ORF1p), a protein encoded by L1, in eight of the EAC cases for which formalin-fixed paraffin embedded tissue was available. With immunohistochemistry, we detected ORF1p in all tumors evaluated. Interestingly, we also observed dim ORF1p immunoreactivity in histologically NE of all patients. In summary, our data show that somatic retrotransposition occurs early in many patients with BE and EAC and indicate that early events occurring even in histologically NE cells may be clonally expanded in esophageal adenocarcinogenesis.