A Three‐Pronged Epigenetic Approach to the Treatment of Hepatocellular Carcinoma
Authors: Ryan A. Hlady, Keith D. Robertson
Journal: Hepatology (2018)

See article on page 1412 Hepatocellular carcinoma (HCC) is among the fastest‐growing causes of cancer‐related death in the United States due to a lack of early detection strategies, increased incidence driven in part by metabolic syndrome, and limited treatment options. Multikinase inhibitors (sorafenib/regorafenib) and immune checkpoint inhibitors (nivolumab) are the three Food and Drug Administration (FDA)–approved drugs for HCC treatment, with only the latter having potential to dramatically improve outcome, emphasizing the unmet need for alternative therapeutic strategies. In this issue of hepatology, Liu et al. explore the role of the DNA methyltransferase (DNMT) inhibitor (DNMTi) SGI‐110 for the treatment of HCC in preclinical models.(1) SGI‐110 (guadecitibine) is a second‐generation DNMTi in which 5‐aza‐2‐deoxycytidine (decitabine) is linked to deoxyguanosine, conferring additional stability and a longer half‐life due to protection from deamination. Global and locus‐specific deregulation of DNA methylation has been attributed to virtually all types of human disease, including cancer. This is particularly important as DNA methylation is a reversible marker that modulates transcription and is a key regulator of development, cell fate, and suppression of endogenous retrovirus (ERV) expression. In the context of HCC, aberrant epigenetic silencing of tumor suppressors (including RASSF1, E‐cadherin, and p16INK4A) has been established for more than a decade.(2) In general, regulatory regions depleted of methylation and nucleosomes are enriched for transcription factor binding sites and promote expression. Aberrant DNA hypermethylation, particularly within enhancers, is coupled with acquisition of nucleosomes, which serve as docking sites for DNMT binding, linking both DNA methylation and nucleosome occupancy in a bidirectional manner to cancer‐associated processes. In this study, Liu et al. treated three HCC cell lines representing different methylation subtypes (as defined by the Cancer Genome Atlas) with SGI‐110, followed by measures of DNA methylation and nucleosome occupancy using the Infinium 450k array–based AcceSssIble assay developed earlier by this group coupled with gene expression profiling.(1) SGI‐110 exposure reduced protein levels of all DNMTs within 3 days, with subsequent genome‐wide demethylation peaking at 10 days, ultimately leading to a sustained inhibition of cell proliferation in all three cell lines. Depletion of either nucleosome occupancy or DNA methylation in promoter regions led to increased gene expression, while increases in DNA methylation or decreases in nucleosome occupancy in gene bodies suppressed transcription. These findings therefore demonstrate that SGI‐110 has prolonged biological effects, hallmarked by widespread loss of DNA methylation and alterations in nucleosome occupancy that occurred either alone or in conjunction with DNA hypomethylation. Interestingly, genes reactivated following SGI‐110 treatment fell into two distinct categories, those whose promoters became hypomethylated (classical mode of action for DNMT inhibition) and those showing methylation‐independent changes linked to increased nucleosome accessibility. Strikingly, 87% of the methylation‐independent changes were linked to targets of polycomb repressive complex 2 (PRC2) subunit enhancer of zeste homolog 2 (EZH2) or its mark H3K27me3, suggesting that application of SGI‐110 leads to re‐expression of PRC2‐targeted tumor suppressor genes independent of DNA methylation alterations. Moreover, DNMT inhibition in these cell lines revealed that ERVs (HERV‐Fc1, HW33438, MLT, and LTC12C) were consistently up‐regulated. ERVs are endogenous viral remnants left behind from ancient retroviral infections, which are typically silenced by DNA methylation. Enhanced immunogenicity through ERV up‐regulation supports the idea that immune checkpoint inhibitors coupled with adjuvant DNMT inhibition, could represent a promising therapeutic strategy for HCC. This study builds on several other interesting observations of SGI‐110 working alone and in concert with chemotherapeutics to suppress HCC. SGI‐110 has been shown to induce hypomethylation in HCC cell lines, with specific effects on tumor suppressor genes including CDKN2A and RUNX3, leading to prevention of HCC progression in preclinical models.(3) The authors postulated that SGI‐110 would be efficacious in advanced HCC, where treatment options are limited. This paved the way for a phase 2 clinical trial involving patients who failed sorafenib treatment (NCT01752933). DNMT inhibition also increases chemosensitivity, as shown in a study where pretreatment of HCC with low‐dose SGI‐110 resulted in sensitization of tumor cells to oxaliplatin.(4) Furthermore, inhibition of either programmed death (PD) ligand 1 (PD‐L1) or DNMT1 leads to sensitization of HCC cells to sorafenib.(5) In HCC, nivolumab recently gained accelerated FDA approval in patients who previously received sorafenib following the CheckMate 040 study (NCT01658878), which demonstrated an objective response rate of 20%.(6) Immune checkpoint inhibitors including nivolumab and durvalumab work through inhibiting interactions between cancer cells and immune cells. PD‐L1 on the surface of the cancer cell binds to PD‐1 on the immune cell, diminishing the immune activity and allowing tumors to evade immune detection. Both nivolumab and durvalumab function by disrupting the interaction between PD‐L1 and PD‐1 (durvalumab binds PD‐L1, nivolumab binds PD‐1). Examination of more than 200 tumors by Calderaro et al. demonstrated that 17% of HCCs express PD‐L1 in 1%‐30% of neoplastic cells and that 76% of HCCs had PD‐L1 expression on inflammatory cells.(7) Expression of PD‐L1 in cancer cells correlated with clinical features linked to poor prognosis including microvascular invasion, poor differentiation, and satellite nodules; and in inflammatory cells a correlation with macrovascular invasion was observed. Combining checkpoint inhibitors with DNMTi in solid tumors has garnered additional traction, in part due to the Stand‐Up‐To‐Cancer initiative, where a potential cooperative link between these agents was revealed.(8) A handful of non‐small cell lung carcinoma patients previously treated with a first‐generation DNMTi achieved complete remission when subsequently treated with a checkpoint inhibitor. This sparked significant interest in using DNMTi to bolster immune checkpoint therapies. Roulois et al.(9) showed in colorectal cancer that DNMTi up‐regulated ERV expression and induced an interferon response, with similar results reported by Liu et al.(1) Low‐dose DNMTi treatment led to production of ERV‐derived double‐stranded RNAs, making cancer cells behave like virally infected cells ("viral mimicry"), thus potentially promoting cancer cell eradication by the immune system. Furthermore, 5‐azacytidine (another first‐generation DNMTi) up‐regulated expression of immune regulatory pathways (particularly interferon‐based mechanisms) and sensitized melanoma cells to checkpoint inhibition in a preclinical model.(10) Clinical trials are currently recruiting to examine the combined effects of durvalumab and SGI‐110 in HCC (NCT03257761). In conclusion, SGI‐110 potentially delivers a three‐pronged epigenome‐based therapeutic "punch" that could be leveraged in HCC treatment: (1) re‐expression of tumor suppressor genes through promoter/enhancer demethylation, (2) reversal of PRC2‐mediated tumor suppressor silencing through decreased nucleosome occupancy, and (3) up‐regulation of endogenous retroviruses to enhance tumor immunogenicity (with PD‐1/PD‐L1 blockade). Alternative strategies like these are critical given the inability to therapeutically target most genetic mutations in HCC. Clearly, this study has limitations, including the use of cell lines that cannot account for intratumor genetic/epigenetic heterogeneity, drug metabolism/bioavailability, and immune influences. Nevertheless, this study shows that DNMTi have multifaceted effects, which need to be better understood for these agents to reach their full therapeutic potential. We eagerly anticipate the results from upcoming DNMTi/PD‐L1 clinical trials. View this article online at wileyonlinelibrary.com. Potential conflict of interest Nothing to report. Author names in bold designate shared co‐first authorship.Figure 1: A three‐pronged, epigenome‐based approach for the treatment of HCC. SGI‐110 mediates DNA demethylation of promoters/enhancers, leading to re‐expression of tumor suppressor genes, and DNA demethylation of endogenous retroviruses, leading to reduced cellular proliferation and priming for immune therapy. An unexpected mechanism for SGI‐110 is its ability to up‐regulate tumor suppressor genes through the DNA methylation–independent mechanism of nucleosome repositioning. Abbreviation: TSG, tumor suppressor gene.