The Emerging Role of Super-enhancers as Therapeutic Targets in The Digestive System Tumors

Digestive system tumors include malignancies of the stomach, pancreas, colon, rectum, and the esophagus, and are associated with high morbidity and mortality. Aberrant epigenetic modifications play a vital role in the progression of digestive system tumors. The aberrant transcription of key oncogenes is driven by super-enhancers (SEs), which are characterized by large clusters of enhancers with significantly high density of transcription factors, cofactors, and epigenetic modulatory proteins. The SEs consist of critical epigenetic regulatory elements, which modulate the biological characteristics of digestive system tumors including tumor cell identity and differentiation, tumorigenesis, environmental response, immune response, and chemotherapeutic resistance. The core transcription regulatory loop of the digestive system tumors is complex and a high density of transcription regulatory complexes in the SEs and the crosstalk between SEs and the noncoding RNAs. In this review, we summarized the known characteristics and functions of the SEs in the digestive system tumors. Furthermore, we discuss the oncogenic roles and regulatory mechanisms of SEs in the digestive system tumors. We highlight the role of SE-driven genes, enhancer RNAs (eRNAs), lncRNAs, and miRNAs in the digestive system tumor growth and progression. Finally, we discuss clinical significance of the CRISPR-Cas9 gene editing system and inhibitors of SE-related proteins such as BET and CDK7 as potential cancer therapeutics.


Introduction
Digestive system tumors including esophageal cancer, colorectal cancer, gastric cancer, pancreatic cancer, hepatic cancer, cholangiocarcinoma, and gallbladder carcinoma are associated with significantly high mortality and morbidity rates [1]. The multistep transformation of normal cells into malignant cells involves genetic and epigenetic alterations that promote the aberrant expression of critical oncogenes and tumor suppressor genes [1,2]. Epigenetic modifications alter cellular plasticity, differentiation, and reprogramming without changing the primary DNA sequence or the genetic code of organisms [3]. Aberrant changes in epigenetic mechanisms regulating DNA methylation, histone methylation and acetylation, expression of noncoding RNAs, and mRNA methylation are associated with the initiation, growth, and progression of digestive system tumors [2,4].
Previous studies have reported the role of super-enhancers (SEs) in digestive system tumors. In the post-genomic era, the cancer research is focused on the dysregulation of transcriptional dysregulation Ivyspring International Publisher mediated by epigenetic modifications in the enhancer, SE, and gene promoter regions of key tumor suppressor and tumor-promoting genes [5,6]. SEs are defined as large clusters of active enhancers in large clusters that are enriched with high levels of transcription factors (TFs), master co-factors, mediator complexes, RNA polymerase II (RNA pol II), and epigenetic modifications [5,7]. SEs regulate biological processes such as cell cycle, oncogenesis, cellular differentiation, immune response, and drug resistance [5][6][7][8]. In this review, we summarized the transcriptional regulatory mechanisms, therapeutic targets, and oncogenes associated with SEs in the digestive system tumors.

Definition and properties of enhancers and super-enhancers
Enhancers are regulatory DNA sequences that enhance target gene transcription by modulating the gene promoter activity through specific TFs in the mammalian cells [9]. Enhancers contain conserved DNA binding sites for the TFs, RNA polymerase, and co-activators [10]. Functional enhancers are also characterized by specific epigenetic modifications such as histone H3 lysine4 methylation (H3K4me1), histone H3 lysine27 acetylation (H3K27ac), and others [11]. The SE regions were first identified by chromatin immunoprecipitation-coupled sequencing (ChIP-seq) analysis of the mediator complex in the mouse embryonic stem cells (ESCs) [5]. The SE regions span dozens of kilobases compared to the typical enhancer (TE) regions, which span only a few dozen base pairs [5]. The SEs are enriched with a higher density of TFs, co-factors, and enhancer-associated epigenetic modifications compared to the TEs ( Figure 1A). Therefore, SEs drive stronger transcriptional activity than the TEs [5,12]. Furthermore, transcriptional coactivators such as cyclin-dependent kinase 7 (CDK7) and bromodomain protein 4 (BRD4) are enriched in the SE regions [13]. SE-driven gene transcription involves cooperative activities of the constituent enhancers. Therefore, deletion of the constituent enhancers significantly alters the SE-driven gene transcription [14,15]. Cohesins are proteins that reduce the spatial distance between the enhancer region and the gene promoter elements [16]. The transcription of SE-driven genes is highly sensitive to disruptions in the cooperative interactions between various SE-related factors because the three-dimensional chromatin architecture of the SE region is altered [16,17].
The SEs are enriched in genomic regions that harbor disease-associated genetic variants and are potentially diagnosis and therapeutic targets. Several SEs involved in human diseases have been characterized by ChIP-seq [18], chromosome conformation capture [19], and DNase I coupled to high-throughput sequencing (DNase-seq) techniques [20].

Biological functions of SEs
SEs are powerful cis-regulatory elements that define cell identity and modulate the maintenance and differentiation of stem cells by regulating the expression levels of pluripotency genes such as OCT4, SOX2, and NANOG [7,21]. Furthermore, the Hippo-YAP signaling pathway modulates the lineage differentiation of mESCs by regulating the formation of SEs [22]. Aberrant SE-related transcription in the cancer cells is associated with increased proportion of cancer stem cells, tumor recurrence, drug resistance, and metastasis [23].
Immunotherapy has emerged as a promising cancer treatment with high efficacy. However, only a subset of patients responed to immunotherapy whereas others show minimal or no response because of variations in the complex tumor immune microenvironment [24]. The immune response of digestive system tumors is also regulated by SEs [25]. A Recent study showed that SEs promote chemoresistance of tumors by aberrant transcription of genes involved in chemoresistance [26]. Additionally, SEs regulate the initiation and maintenance of chemoresistance in multiple tumors [6,27]. Furthermore, the sensitivity of chemotherapy can be restored by small molecule epigenetic inhibitors of SEs [6].

Characteristics of SEs
The master TFs in the SE region form an interconnected core regulatory circuits, which orchestrates the transcriptional programs in both normal and malignant cells ( Figure 1B) [28]. The transcriptional program in the human ESCs involves collaboration between OCT4, SOX2, and NANOG, which form the core regulatory circuitry in the SE region with auto-regulatory and feed-forward loops [29]. Similarly, ELF3, EHF, and TGIF1 form the interconnected core regulatory circuitry in the SE region of lung adenocarcinoma cells and promote transcription of the three master TFs and their related genes in tandem [30].
SEs also indirectly regulate the biological functions of cancer cells by modulating the expression levels of noncoding RNAs (ncRNAs) [31], including microRNAs (miRNAs) [32], long noncoding RNAs (lncRNAs) [33], and circular RNAs (circRNAs) [34]. For example, knockdown of SE-associated lncRNA CCAT1-L significantly decreasing the MYC transcript levels by reducing the long-range interactions between MYC promoter and its enhancers [35]. Enhancer RNAs (eRNAs) are ncRNAs that are transcribed from the DNA sequences in the SE regions and are involved in the regulation of gene expression, RNA splicing, translation, and epigenetic mechanisms [34,36]. The eRNAs regulate initiation, proliferation, apoptosis, migration, adhesion, drug resistance, and immune response of multiple tumors [37,38]. Moreover, eRNAs promote transcription by enhancing the formation of SE-gene promoter loops ( Figure 1C) [39]. Gene expression is regulated by the phase-separated multi-molecular assemblies of transcriptional protein complexes in the SE region [17]. In the liquid-liquid phase separation (LLPS) model, SEs display a transcriptional bursting pattern by forming chromatin loops with their target gene promoters ( Figure 1D) [40]. Transcriptional co-activators BRD4 and MED1 are enriched in the SEs and form phase-separated droplets, which contributes to the compartmentalization and concentration of the transcriptional machinery from the nuclear extracts at the SEs [41]. The intrinsically disordered regions (IDRs) associated with the SE-enriched transcriptional co-activators play an important role in the process of LLPS through self-associating electrostatic, polar, and hydrophobic interactions [40,42,43]. For example, LLPS of NUP98-HOXA9 promotes transcriptional activation of the tumor genes by forming a broad super-enhancer-like binding patterns [44].

Oncogenic roles and regulatory mechanisms of SEs in the digestive system tumors
SEs play a critical role in the development of multiple digestive system tumors (Figure 2). Genome-wide profiling of tumor tissues identified SE-associated oncogenes, PHF19 and TBC1D16 [45]. Silencing of SE-related oncogenes significantly inhibit the tumor progression [45]. SEs activate several oncogenes and signaling pathways in esophageal adenocarcinoma (EAC) [46]. Four EAC-specific master regulatory transcription factors, ELF3, KLF5, GATA6, and EHF form interconnected regulatory loops, which co-operatively activate the expression levels of all the four TFs [47]. These master TFs occupy most EAC-related SEs and promote the survival and proliferation of EAC cells through cooperative transcription of the EAC-related genes [46]. In the following section, we discuss the oncogenic roles of SEs and the underlying mechanisms that regulate the biological processes in different digestive system tumors ( Table 1).

SE participate in the transcription reprogramming of oncogenes
SEs demonstrate stronger transcriptional activity than the TEs [5]. Cancer-related SEs promote malignant tumor progression by increasing the transcription levels of the oncogenes. For example, binding of MYC to the active elements in the SE region drives overexpression of HOXB8 during colorectal cancer (CRC) progression [48]. In some case, reorganization of SEs due to chromosomal rearrangements results in enhancer hijacking and is associated with the activation of oncogenes. In CRC, IGF2 is a target of enhancer hijacking and is activated by the formation of a 3D contact domain that involves tandem-duplicated IGF2 and the lineage-specific SE [49]. In several cases, SE-driven transcription is associated with tumor malignancy phenotypes. For example, SE-driven EVI1 is a key promoter of oncogenic transcription remodeling and is associated with the aggressive malignant phenotype in pancreatic ductal adenocarcinoma (PDAC) [50]. Similarly, SE-driven TGFBR2 transcription promotes pancreatic cancer growth and progression via TGF-β signaling [51,52]. SE-mediated transcriptional activation regulates expression levels of several oncogenes. The overexpression of ZFP36L2 promotes growth and malignancy of gastric cancer (GC) cells, and its transcription levels are regulated by a tandem duplication in the SE region [53]. Interleukin-6 (IL-6) is overexpressed in pancreatic cancer. Bao et al. demonstrated that genetic deletion of IL6-SEa or treatment with two inhibitors of the SE-related BET protein, JQ-1 and I-BET762, significantly reduced the IL-6 expression levels in multiple cancer cells [54].
Cancer cells adapt to the dynamic tumor microenvironment through transcriptional reprogramming, which is significantly associated with tumor metastasis and recurrence. In ESCC, transcriptional reprogramming during tumor progression and metastasis involves gain or loss of several thousand enhancers and alterations in hundreds of putative SEs [55]. Transcriptional reprogramming mediated by SEs also regulates CRC development and progression. Inhibition of KDM6 induces global enhancer reprogramming, especially in the SE-associated genes such as ID1 that control stemness of CRC cells [56]. Liver metastasis in CRC patients is also induced by transcriptional reprogramming. Liver-specific TFs such as FOXA2 and HNF1A bind to altered TEs and SEs in the CRC cells and activate liver-specific gene transcription that drives liver metastasis [57]. Furthermore, HNF1A is overexpressed and HNF1Abinding motifs are enriched in the SEs during liver metastasis in CRC [58]. Transcriptional reprogramming mediated by SEs also promotes HCC progression. SIRT7 SE is an acquired SE in nonalcoholic fatty liver disease-related HCC that drives metabolic reprogramming and promotes oncogenic activity through SIRT7-mediated chromatin deregulation of tumor-suppressor genes [59]. Xiao et al. performed single-cell holo-transcriptome sequencing and demonstrated genome-wide SE remodeling during the malignant transformation of HCC cells based on data from the [60]. In another study, micro-scale chromatin profiling of primary GC tissues showed genome-wide reprogramming of the SE landscape and dense co-occupancy of transcription factors CDX2 and HNF4α, which promote aberrant expression of oncogenes [61]. These data demonstrate that understand the mechanisms of SE-mediated transcription and reprogramming of oncogene transcription provide more therapeutic strategies for targeting digestive tumors.

SE-driven oncogenes promote proliferation and metastasis of cancer cells
SEs are potential targets for inhibiting the expression levels of SE-related oncogenes to reduce tumor cell proliferation and migration. In a subset of esophageal squamous cell carcinoma (ESCC), interactions between the bromodomain protein BRDT and the △Np63 transcription factor in the SE regions of squamous phenotype-related genes such as KRT14, FAT2, and PTHLH increase their transcription levels, thereby promoting ESCC cell migration [62]. Therefore, preferential degradation of BRDT by MZ1 reverses the activation of these oncogenes [62]. Se-driven AJUBA is activated by TCF4 and regulates migration and invasiveness of HCC cells [63].
Differential H3K27Ac marks were identified in the enhancer regions of SE-driven oncogenes such as c-MYC, MED1, OCT4, NANOG, and SOX2 in the PDAC cell lines [64]. However, treatment with GZ17-6.02, a cocktail of natural anticancer agents, reduced progression of PDAC cells by decreasing the H3K27ac levels in the hyperacetylated SE regions of these oncogenes [64]. Furthermore, triptolide, a bioactive compound extracted from the Chinese herb Tripterygium wilfordii, inhibits proliferation and migration of PDAC cells by disrupting SEs and downregulating the expression levels of SE-driven genes such as BRD4, MYC, and RNA pol Ⅱ [65].

Tumor diagnostic and prognostic biomarkers
In general, high expression levels of SE-related oncogenes are associated with poor prognosis of cancer patients [66]. Huang et al.reported that a prognostic signature of six enhancer-associated differentially expressed genes (DEGs) showed superior performance in predicting the survival outcomes of HCC patients [67]. The co-operative interaction between DHX37 and PLRG1 in the promoter and SE regions induces transcription of cyclin D1, and promotes proliferation of HCC cells; high cyclin D1 expression is associated with poor prognosis of HCC patients [68]. These data demonstrate that SE-induced expression of oncogenes and tumor suppressor genes is associated with the diagnosis and prognosis of digestive system tumors and may contribute to the development of precision medicine.

Regulation of immune evasion
Immune evasion is an important mechanism that facilitates tumor cell survival, growth, and metastasis. Recent studies have shown the relationship between SEs and the tumor immune escape mechanisms. The immune checkpoint blockade proteins, programmed cell-death protein 1 (PD-1) and programmed cell-death 1 ligand 1 (PD-L1), play an important role in determining the tumor immune microenvironment and anti-cancer immune response [69]. The synchronous expression of PD-L1 and PD-L2 is driven by the PD-L1L2-SE in the tumor cells; inhibition of PD-L1L2-SE significantly reduces the expression levels of PD-L1 and PD-L2 and increases the lethality of the cytotoxic T cells [70]. CD47 is expressed on the surface of macrophages and other immune cells and inhibits phagocytosis by binding to the SIRPa receptor [71]. In breast cancer, CD47 expression is upregulated by a CD47-associated SE [72]. The upregulation of SE-regulated IL-20RA is associated with increased immune evasion of CRC cells through oncogenic and immune response pathways that reduce infiltration of N1 neutrophils, M1 macrophages, and T cells [25].

Noncoding RNAs associated with SEs in digestive system tumors
In addition to regulating the expression of protein-coding genes to directly regulate biological processes, SEs indirectly regulate the biological functions of cancer cells by activating the expression of ncRNAs such as miRNAs, eRNAs, and lncRNAs [31]. In the following, we discuss the roles and mechanisms of these SE-associated ncRNAs in tumor progression.

MicroRNAs
SEs modulate tumor malignancy by regulating the tissue-specific expression levels of various miRNAs. For example, the SE of KLF6 regulates the proliferation of HCC cells by upregulating p21 and p53, whereas deletion of KLF6 SE inhibits HCC proliferation via miR-1301 overexpression [73]. MiR-122 is another SE-driven liver-specific miRNA that is highly expressed in the newborn and adult liver tissues. MiR-122 participates in HCC growth and progression by directly targeting the regulators of Hippo signaling pathway [74].

LncRNAs
LncRNAs are noncoding RNAs that are longer than 200 nucleotides. The dysregulation of lncRNAs is associated with tumorigenesis. SE-associated lncRNAs such as HOTAIR, XIST, SNHG5, and LINC000940 regulate the expression of ESCC hallmark genes [85]. The binding of TCF3 and KLF5 transcription factors to the SE region induces the expression of LINC00094, which promotes growth and survival of ESCC cells [85]. TP63 transcription factor induces SE-dependent transcription of LINC01503, which subsequently promotes proliferation, migration, and invasion of squamous cell carcinoma cells via the MAPK and Akt signaling pathways [86]. In ESCC cells, lncRNA CCAT1 is upregulated through the binding of master transcription factors such as TP63, SOX2, and KLF5 to the SE region; CCAT1 overexpression drives ESCC proliferation and progression [86][87][88].
SE-associated lncRNAs also regulate CRC growth and progression by modulating the expression level of several CRC-related genes [89]. The overexpression of SE-regulated lncRNA RP11-569A11.1 inhibits CRC malignancy by increasing IFIT2 expression levels [89]. Conversely, the overexpression of SE-regulated lncRNA AC005592.2 promotes CRC progression [90]. In CRC tissues and cells, MYC transcription is regulated via long-range chromatin looping induced by the SE-regulated lncRNA CCAT1-L, which is transcribed 515 kb upstream of the MYC promoter [35]. Furthermore, transcription levels of dysregulated CRC-related lncRNAs are directly regulated by the 5-hydroxymethylcytosine (5hmC) levels in the genomic loci or through aberrant activities of 5hmCmodified TEs, SEs and promoters [91].
LncRNAs are novel candidate oncogenes and potential diagnostic biomarkers for HCC. The overexpression of SE-regulated lncRNAs HCCL5 promotes survival, migration, and EMT of HCC cells, and correlates with poor survival outcomes of HCC patients [38]. LncRNA-DAW promotes HCC cell proliferation by activating the Wnt/β-catenin pathway [92]. Overall, the above findings suggest that SEs and SE-regulated ncRNAs play a critical role in the progression of digestive system tumors through epigenetic regulation and modulation of oncogenic signaling pathways.

Targeting super-enhancers as a therapeutic strategy in digestive system tumors
Specific inhibition of SEs formation and SE-dependent activation of oncogene transcription represents a novel strategy for cancer therapy. Inhibitors targeting SE components bromodomain and extra terminal domain (BET) or cyclin-dependent kinase (CDK), have shown immense potential in cancer therapy and are currently undergoing clinical trials to determine the efficacy and safety in treating patients with digestive system tumors ( Table 2). Furthermore, disruption of the formation and structure of the oncogenic SEs by gene editing technology has emerged as a new potential novel strategy to treat cancers (Figure 3).

BET inhibitors
The BET protein family members such as BRD2, BRD3, BRD4, and BRDT are important regulators of epigenetic modifications and gene transcription [93]. Among these, BRD4 occupies the SE regions and regulates gene transcription by binding to H3K27ac [94]. BRD4 inhibition disrupts the communication between SEs and their target promoters, and significantly decreases the expression of SE-driven oncogenes, which are essential for cancer progression [95].
Currently, the clinical efficacy of BET inhibitors is being evaluated for various cancers. JQ1 is the most widely studied small-molecule BET inhibitor that blocks SE-induced oncogene transcription [96]. JQ1 inhibits proliferation of CRC cells by disrupting Cmyc-SE and downregulating c-MYC expression [97]. The cell cycle progression and survival of BRAF V600E -mutant CRC cells was reduced by treatment with a combination of JQ1 and vemurafenib, a selective inhibitor of mutated BRAF [98]. KDM6A-deficient pancreatic cancer cells show aberrant activation of SEs that regulate oncogenes such as △Np63, MYC, and RUNX3, but are but are highly sensitive to BET inhibitors [99].   The clinical efficacy and safety profiles of BET inhibitors including I-BET762 and I-BET151 are currently being evaluated in patients with digestive system tumors. I-BET762 inhibits PDAC cell proliferation and progression by inducing G0/G1 phase cell cycle arrest and cell death [100]. I-BET151 inhibits the growth of CRC cells by downregulating the expression of SE-driven IL-20RA [25]. However, some clinical trials have reported adverse effects of BET inhibitors, including moderate gastrointestinal toxicity and fatigue, thrombocytopenia, and cardiotoxicity [101][102][103]. Androgen receptor (AR)-dependent transcription in prostate cancer cells is suppressed by the second-generation BET inhibitor ABBV-744, which selectively inhibits the BD2 domain of BET family proteins and displaces BRD4 from SEs with AR [104]. The adverse effects of traditional BET inhibitors can be overcome by proteolysis-targeting chimeras, which effectively inhibit solid tumors with low cytotoxicity by degrading the BET proteins [105].
Several solid tumors demonstrate resistance against BRD4 inhibitors. Wang et al. demonstrated that BRD4 phosphorylation at tyrosine 97/98 induced resistance against the BET inhibitors in CRC Cells [106]. Furthermore, because methylation status of the promoter region regulates gene expression, drugs targeting epigenetic modifications are also being evaluated for cancer therapy [107].

CDK inhibitors
CDKs regulate cell cycle and transcription by controlling the initiation and elongation of RNA pol Ⅱ [108,109]. Selective inhibition of CDK7 significantly reduces transcription of oncogenic TFs, especially those associated with SEs [110]. Therefore, inhibition of CDK7 is a promising therapeutic strategy against digestive system tumors.
THZ1 is a covalent CDK7 inhibitor that inhibits RNA pol Ⅱ C-terminal domain phosphorylation [108]. This decreases the number of RNA pol Ⅱ molecules in the SE region and inhibits the transcription of SE-related oncogenes [94]. Low-dose THZ1 significantly reduces SE-driven transcription of PAK4, RUNX1, DNAJB1, SREBF2, and YAP1 oncogenes in the ESCC cells [111]. THZ1 also suppresses the growth of ESCC cells by downregulating the expression of lncRNA LINC00094 [85]. Huang et al. [112] showed that nanoparticles with high drug loading of JQ1 and THZ1 (J/T@8P4s) effectively suppressed SE-associated oncogenic transcription in the PDAC cells. In the in vitro and in vivo HCC models, THZ1 exerts significant antitumor effects by selectively suppressing the expression levels of SE-associated oncogenes [113]. SY-1365 is a highly potent and selective CDK7 inhibitor that inhibits the growth of various cancers in both in vivo and in vitro experimental models and is currently undergoing clinical trials [110]. CDK7/9 inhibitor SNS-032 significantly inhibits the growth of ESCC cells and xenografts [114]. Therefore, inhibition of CDK7 and CDK9 is a promising therapeutic strategy for digestive system tumors.

Targeting oncogenic SEs by gene editing
In recent years, gene-editing technology has emerged as a convenient tool to study the functions of genes by correcting the mutated genes or modifying the DNa sequences of genes in human diseases. The CRISPR-Cas9 system is the most efficient gene-editing tool that is used for basic and clinical research. Gene-editing technology is also used to design therapeutics targeting oncogenic or disease-related SEs.
CRISPR-Cas9 technology can be used to prevent the formation of disease-related SEs which are generated by genetic mutations such as base insertions, deletions, or substitutions and chromatin rearrangements. It has also been used to investigate mechanisms related to tumorgenesis and identify new drug targets [115]. CRISPR-Cas9 technology can be used to study the cooperation between individual enhancers within the SE region by knocking out individual enhancers [116]. Moreover, the CRISPR-Cas9 system can also alter the functions of SEs by deleting specific DNA sequences and promoting nonhomologous recombinant repair [117]. For example, disruption of the SE regions in the RUNX1 gene by the CRISPR-Cas9 system induces apoptosis of leukemic cells [118]. Moreover, the combination of advanced genome-wide sequencing and CRISPR-Cas9 technology is used to investigate the in vivo functions of SEs in the mammary gland genome [119]. This strategy may provide new insights into investigating SEs-associated tumorigenesis in the future.
CRISPR-Cas9 technology has also been used to study the function of SE components in the digestive system tumors. Deletion of the TP63 promoter or individual components of the TP63 SE through CRISPR-Cas9 decreases the expression levels of TP63 and inhibits the proliferation of the ESCC cells, thereby indicating the association of TP63 with tumor malignancy [87]. Furthermore, CRISPR-Cas9mediated knockdown of the SE complex components such as CDK7, BRD4, EP300, and MED1 significantly inhibited the proliferation of HCC cells [113]. The CRISPR-Cas9 system can also be used to alter SE-driven oncogene expression through single nucleotide polymorphisms (SNPs), which alter the gene expression patterns by disrupting the formation and interaction of the SEs [120]. H3K4me1 and H3K27ac markers are enriched in the SE region of SNP rs6854845, which is a risk factor associated with colon cancer [121]. Genome editing by CRISPR-Cas9 alters the transcription of genes by modulating the interactions between SE-associated genes and SNPs [121].
Although CRISPR-Cas9 is a highly efficient and convenient tool, it has drawbacks. In some cases, ineffective base editing and off-target effects limit the application of CRISPR-Cas9 technology [122]. Therefore, the efficacy and safety of gene-editing technology in targeting disease-related SEs requires careful characterization for clinical applications. Li et al. developed a more efficient enCRISPRa system for enhancer activation and the enCRISPRi system for enhancer inhibition to analyze the in situ and in vivo functions of enhancers [123]. Liu et al. used the CRISPR affinity purification in situ of regulatory elements (CAPTURE) proteomics approach to identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions without bias [124]. This proteomics approach captures protein complexes that bind to SE with greater sensitivity and specificity and can be used to find new targets to develop small-molecule inhibitors for cancer therapy. A new precise gene-editing tool, Prime Editor, has been developed to edit SE formation and overcome the non-specific base editing and off-target effects of the CRISPR-Cas9 system [125]. Overall, there is great scope for the application of gene-editing tools in SE editing for cancer therapy.

Conclusion
The aberrant activity of SEs drives oncogene transcription in digestive system tumors and promotes tumor cell proliferation, migration, and malignancy. SE-associated miRNAs, eRNAs, and lncRNAs also promote growth and progression of digestive system tumors indirectly. SEs are cell-type-specific. Hence, they are potential biomarkers for identifying and classifying different subtypes of digestive system tumors. However, further studies are needed to determine frequently dysregulated SEs and the downstream oncogenic signaling pathways in different digestive system tumors.
SEs are promising therapeutic targets in digestive system tumors. However, the potential target of gene editing at the DNA level is still unclear because SEs occupy large regions of DNA. At present, SE-targeted therapy is focused on small-molecule inhibitors of BET and CDKs, which have shown significant antitumor efficacy in several pre-clinical studies and are currently undergoing clinical trials ( Table 2). However, the clinical application of BET and CDKs inhibitors are limited because they interfere with the transcription of many proteins and cause significant toxicity and adverse events. SY-1365 is a selective CDK7 inhibitor that entered the phase I clinical trial in 2017 [126]. However, preliminary findings demonstrate that the efficacy of SY-1365 is average. Therefore, effective CDK7-targeted anticancer therapyrequires further verification in the future. Moreover, further investigations are necessary to determine the efficacy and safety profiles of SE-targeting inhibitors in combination with other antitumor agents such as chemotherapy drugs and anti-tumor monoclonal antibody drugs in advanced or drug-resistant digestive system tumors.
Several studies have demonstrated that CRISPR-Cas9 gene-editing technology can be used to abolish SE formation in cancer cells. This modern technology shows great promise for individualized treatment of cancer patients based on the status of many SEs. In conclusion, SEs play important roles in digestive system tumors by driving oncogenic expression and oncogenic signaling pathways. Further studies are necessary to unravel the complexity of SE-related mechanisms, their clinical significance as therapeutic targets, and investigate more effective therapies targeting SE.