The epitranscriptome of high-grade gliomas: a promising therapeutic target with implications from the tumor microenvironment to endogenous retroviruses

Glioblastoma (GBM) comprises 45.6% of all primary malignant brain cancers and is one of the most common and aggressive intracranial tumors in adults [1]. Due to its highly aggressive nature and inevitable recurrence, its disease course has one of the highest mortalities, with a 5-year survival rate of only 5% despite maximal surgical resection and adjuvant chemoradiation [2].

GBM is a highly heterogeneous neoplasm with a wide range of proteomic, genetic, and epigenetic dysregulation [3]. Many enzymes responsible for regulating protein and DNA modifications are currently targets of cancer trials and therapies. However, studying RNA modifications, or RNA epitranscriptomics, is a new frontier within neuro-oncology [4]. Simply put, epitranscriptional modifications include dynamic, covalent modifications that can affect the stability, translation, and function of RNA. Although eukaryotic RNA modifications have been known since the 1970s, most studies focused on transfer RNA (tRNA) and ribosomal RNA (rRNA). Only within the last decade has there been extensive characterization of modifications of mRNA and various non-coding RNAs. RNA modifications have steadily moved towards the limelight as scientists appreciated that RNA does not only act as an effector molecule (tRNA and rRNA) or intermediate in protein synthesis (mRNA) but also directly affects gene expression via non-coding RNAs such as microRNA (miRNA) and long ncRNA (lncRNA) [5‐7].

Few dedicated studies of posttranscriptional modifications and their potential as therapeutic targets occurred initially. Still, in the past few years, this field has become an active field of investigation for brain tumors [4]. Several lines of evidence suggest that epitranscriptomic dysregulation contributes to glioma pathogenesis [4, 8‐10]. The three major epitranscriptomic players implicated in both physiologic regulation and disease include “writers” that add a specific modification, “erasers” that remove a specific modification, and “readers” that identify and bind modified nucleotides.

A common consequence of pretranscriptional (i.e., epigenetics) and posttranscriptional (i.e., epitranscriptomic) dysregulation is the activation of transposable elements (TEs). Interestingly, in the last decade of cancer research, a form of TEs known as endogenous retroviruses (ERVs) has been shown to play a dichotomous role in driving oncogenesis and serving as potential antigens for immunotherapy [11]. First described in 1981, human ERVs (hERVs) are relics of retroviral infection of the ancestral germline. hERV genes account for nearly 9% of the human genome and are transcriptionally silent in normal cells. hERV expression is associated with many cancers. hERV expression also is related to autoimmune and neurodegenerative diseases. Augmenting hERV inflammatory effects through a cellular state known as “viral mimicry” is being explored to sensitize tumors to immunotherapy [12]. Viral mimicry enhances hERV expression by reversing silencing marks that suppress hERV gene expression. The fundamental premise is that a transient increase in hERV expression may induce an innate antiviral and adaptive immune response that sensitizes and homogenizes tumor cells for immunotherapy.

Recent publications extensively reviewed how RNA modifications participate in cancer and glioma development/progression, regulate the tumor microenvironment (TME), and encourage the development of drug resistance [4, 9]. This review aims to provide background information about epitranscriptomics and discuss the therapeutic potential of modulating epitranscriptomics in high-grade gliomas and GBM. We will also further discuss the rationale for using a hERV-mediated viral mimicry strategy as part of a combination immunotherapy for GBM.

The role of RNA modification in cancers is incredibly complex and challenging to study, primarily due to limitations in sequencing technologies. Conventional sequencing techniques such as bulk RNA-seq and scRNA-seq quantify and normalize for mRNA expression but are not specifically developed to detect RNA modifications. Taguchi et al. recently reviewed developments in epitranscriptome spatial detection and data analysis [41]. Advancements in de novo sequencing will be discussed later in this review.

RNA modifications, namely m6A methylations, have been found to influence the tumor microenvironment (TME), immune cell-infiltrating characteristics, and, ultimately, patient outcomes [4, 42]. RNA modification patterns are particularly clinically relevant since they may portend responses to immunotherapy [42]. Similarly, Lin et al. utilized the TCGA-GBM and TCGA-LGG databases to explore patterns of differentially expressed m6A regulators in glioma [43]. Using principal component analysis, two glioma subgroups were identified, and one cluster was found to have a worse prognosis, higher WHO classification grade, and higher immune infiltration. Based on the combination of m6A RNA methylation and the landscape of the immune microenvironment, a novel 4-gene (TAGLN2, PDPN, TIMP1, EMP3) prognostic model was developed and validated with a good prediction. In addition, PDPN and TIMP1 were found to be highly expressed in high-grade glioma via The Human Protein Atlas database, and both correlated with m6A and macrophage marker CD68 in glioma tissue samples, which may serve as potential biomarkers for glioma prognosis.

Molecular alterations characterizations such as isocitrate dehydrogenase IDH1/2 mutations, 1p/19q codeletion, and MGMT promoter methylation, have improved the accuracy of diagnostics, prognostics, and prediction of treatment response for glioma patients. However, we have yet to observe any tangible improvement in either clinical management or patient outcomes. A potential utility of epitranscriptomics is to be used in conjunction with known glioma sub-entities to further advance tumor stratification and help guide treatment selection [15]. According to studies mining the Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and Repository for Molecular Brain Neoplasia Data (REMBRANDT), epitranscriptomic regulators appear to be differentially expressed based upon molecular alterations and clinical attributes. For example, METTL3, FTO, and YTHDC1 are significantly differentially expressed between IDH-mutant and IDH-wildtype high grade gliomas [44]. Additionally, m6A writer METTL3 correlates with poor OS in IDH-wildtype but not in IDH-mutant gliomas [45].

A critical limiting factor of most epitranscriptomic cancer studies has been the scientific indifference towards RNA modifications other than m6A. Given the integrated process of interactions between different RNA methylation modifications, studying a single type of RNA modification in tumors may not comprehensively elucidate epitranscriptomic effects. A recent study by Li and colleagues recognized this. It developed a GBM score’ based on the differentially expressed genes (DEGs) between groups showing RNA modification patterns of writers that catalyze m1A methylation, m6A methylation, APA, and A-to-I RNA editing [46]. Out of the 26 writers analyzed, 15 were found to be more highly expressed in tumor tissue than adjacent normal tissue. Using Spearman’s correlation analysis, positive correlations occurred among several writers, which indicate that crosstalk among RNA modification writers may mediate the formation of distinct RNA modification patterns and GBM progression. After consensus clustering analysis, two clusters emerged, with one having a significantly shorter survival. In this cluster with higher mortality, GSVA was used to estimate the signaling pathways with gene set enrichment. Signaling pathways found to be significantly enriched were those involved in NK cell-mediated cytotoxicity, Toll-like receptor activation, JAK-STAT, and chemokine signaling. Additionally, using CIBERSORT, this same cluster was found to have higher levels of immunosuppressive cells such as M2 macrophages. Of note, several RNA modification writers, including RBM15, RBM15B, TRMT6, CLTP1, PABPN1, ADARB1, and CPSF1, were found to be positively associated with M0 macrophage differentiation.

The immunosuppressive tumor microenvironment has been identified as the primary reason for the failure of immune therapy in GBM patients; currently, there are no FDA-approved immunotherapies for GBM [47]. Accumulating evidence has shown a strong correlation between RNA modifications, namely m6A, and immunomodulation, and thus the efficacy of immunotherapies in GBM patients [43, 46, 48‐50]. This correlation does not appear to be unique to primary brain tumors, as similar data has been found in colorectal cancer [51], colon cancer [52], gastric cancer [42], bladder cancer [53], lung adenocarcinoma [54], and head and neck squamous cell carcinoma [55]. This collection of recent investigations indicates that RNA modification shapes the complexity of the TME and its response to immunotherapy. Han et al. provide a more complete review of this field [56]. While exciting, extensive work is needed to understand modifications other than m6A, crosstalk between modifications, and cell types modulated by RNA modification. To fully understand the underlying mechanisms and therapeutic potential, further in vitro and in vivo studies are needed using meticulous methods to dissect RNA modifications and their regulators in tumor cells versus immune cells [56].

Epitranscriptomic sequencing technologies may be divided into four fundamental approaches: antibody-based, reverse-transcription signature-dependent, enzyme-dependent, and chemically assisted [8]. Antibody-based methods are the most widely used modality for m6A, m5C, ac4C, and m7G modifications; however, drawbacks include limited specificity, lack of stochiometric outputs, and high input material requirements. Reverse transcription signature-dependent techniques rely on insertion-deletion mutations (indels), mismatches, and truncations at the modification sites. Still, they are prone to false positives due to errors in library preparation and sequencing or single-nucleotide polymorphisms [57, 58]. Enzyme-dependent sequencing technologies rely on various enzymes such as demethylases, endonucleases, and exonucleases to discriminate regular bases from modified ones [8]. The efficiency and sequence biases of the respective enzyme characterize its limitations. Lastly, chemical-assisted technologies combine chemical treatments with next-generation sequencing [8]. Recent chemical methods have addressed the gap between the dynamics and reversibility of RNA modifications by allowing absolute quantification at single-base resolution for m6A, pseudouridine, m5C, and ac4C.

Novel sequencing technologies that allow simultaneous detection of multiple modifications are vital to understand the crosstalk between modifications further and drive the field of epitranscriptomics. Third-generation methods such as SMRT and Nanopore sequencing hold promise but have faced challenges, including limited signal-to-noise ratios, complex algorithms, and high error rates and costs [59]. In addition, spatial epitranscriptomic elucidation of the mRNA modification profiles of individual cells in both spatial and temporal dimensions is likely to become an important area in developmental and cancer biology [8, 60].

As occurs in pluripotent embryos, many cancers are known to reactivate transposable elements (TE) through chromatin remodeling, DNA hypomethylation, and histone modifications [12]. TEs are fundamentally heterogeneous, comprising two main types: DNA transposons and retrotransposons, which include Long Interspersed Nuclear Elements (LINEs), Short Interspersed Nuclear Elements (SINEs), and Long Terminal Repeat (LTR) Retrotransposons. Our focus in this review will be on LTR retrotransposons or human ERVs (hERVs).

The genome of an intact hERV provirus comprises at least 5’ and 3’ long terminal repeats (LTRs) flanking an internal Gag (group-specific antigen)-Pro (protease)-Pol (polymerase) polyprotein-coding sequence. Gag is cleaved by Pro to generate a virus-like particle that contains the fusion protein and ERV mRNA. hERVs may also contain a remnant envelope (Env) and other accessory genes but are generally not infectious [12]. HERV-K (HML-2), first reported in 1986, is the most recent hERV integrated into genomic DNA. Unlike other hERVs, HERV-K contains a near-full-length transcript in the human genome that includes open reading frames (ORFs) of HERV-K Gag, Pol, and Env, which can be read and translated into functional retroviral proteins [61].

While necessary for mammalian embryologic development, ERV activation and transposition can compromise host health secondary to aberrant transcriptional regulation; thus, constant surveillance is needed to maintain homeostasis [62]. Although the ERV regulatory network mechanisms are poorly understood, recent advancements have helped elucidate how mammals effectively regulate ERV expression.

ERV regulatory networks fundamentally consist of pre-transcriptional and post-transcriptional regulatory networks [63]. Pre-transcriptional regulators include zinc-finger proteins (ZFPs), TRIM28-SETDB1, human silencing hub (HUSH) complex, SWItch-sucrose non-fermentable (SWI-SNF) complex, MORC proteins, Lymphoid-specific helicase (LSH), and P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs). Alternatively, piRNAs, nuclear exosome targeting (NEXT) complex, and RNA methylation comprise the post-transcriptional modulators. Table 2 summarizes the proteins and function of the ERV expression regulators.

Several drugs, such as 5-azacytidine and decitabine inhibiting DNA methylation, are FDA-approved for hematological tumors. Of the many similarities and parallels between DNA and RNA methylation, the most similar is m5A. Interestingly, studies show the clinical response to hypomethylating therapies do not correlate with DNA methylation status, and approximately 90% of 5-azacytidine is incorporated into RNA [103]. Thus, these therapies’ antiproliferative effects, either RNA- or DNA-mediated, and drug-repurposing potential remain hotly debated, with further mechanistic studies needed.

Though no FDA-approved drugs currently target epitranscriptomic regulators, there is preclinical evidence of the anti-tumor effects of RNA-modifying therapy and a growing cadre of interested biotech companies [103]. Most small molecule inhibitors for RNA-modifying therapy have been developed against m6A regulators. For example, the METTL3–METTL14 catalytic activity inhibitor STM2457 has been validated in vitro and in vivo for hematological tumors [9]. Additionally, inhibitors against FTO, ALKBH5, IGF2BP1, and ADAR1 have shown promising anti-tumor properties in vitro and in vivo [104]. However, as mentioned previously, these inhibitors will likely require chemical modification to improve their BBB penetration for glioma treatment.

Utilizing glioblastoma stem cells (GSCs) and xenograft mice, MA2, the ethyl ester form of meclofenamic acid (MA), an FDA-approved non-steroidal anti-inflammatory drug, was identified as a selective inhibitor of FTO that increases m6A mRNA levels and suppresses GSC-initiated tumor progression [105, 106]. Similarly, small-molecule compounds inhibiting pseudouridine synthase 7 impair GSC growth in vitro and, in mouse xenografts, improve prognosis [107]. Lastly, inhibition of TYK2, a mediator of both PUS7- and ADAR1-containing pathways in GSCs, demonstrated impaired GSC self-renewal and stemness [108]. As these molecules enter clinical trials, how they affect the landscape of cancer therapies targeting gene expression dysregulation and impact GBM patient outcomes will be exciting.