The double-edged sword of (re)expression of genes by hypomethylating agents: from viral mimicry to exploitation as priming agents for targeted immune checkpoint modulation

Abstract

Hypomethylating agents (HMAs) have been widely used over the last decade, approved for use in myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). The proposed central mechanism of action of HMAs, is the reversal of aberrant methylation in tumor cells, thus reactivating CpG-island promoters and leading to (re)expression of tumor suppressor genes. Recent investigations into the mode of action of azacitidine (AZA) and decitabine (DAC) have revealed new molecular mechanisms that impinge on tumor immunity via induction of an interferon response, through activation of endogenous retroviral elements (ERVs) that are normally epigenetically silenced. Although the global demethylation of DNA by HMAs can induce anti-tumor effects, it can also upregulate the expression of inhibitory immune checkpoint receptors and their ligands, resulting in secondary resistance to HMAs. Recent studies have, however, suggested that this could be exploited to prime or (re)sensitize tumors to immune checkpoint inhibitor therapies. In recent years, immune checkpoints have been targeted by novel therapies, with the aim of (re)activating the host immune system to specifically eliminate malignant cells. Antibodies blocking checkpoint receptors have been FDA-approved for some solid tumors and a plethora of clinical trials testing these and other checkpoint inhibitors are under way. This review will discuss AZA and DAC novel mechanisms of action resulting from the re-expression of pathologically hypermethylated promoters of gene sets that are related to interferon signaling, antigen presentation and inflammation. We also review new insights into the molecular mechanisms of action of transient, low-dose HMAs on various tumor types and discuss the potential of new treatment options and combinations.

Keywords: Acute myeloid leukemia; Azacitidine; Cancer; DNA methylation; Decitabine; Endogenous retroviral elements; Hypomethylating agents; Immune checkpoint blockade; Immune checkpoint inhibitors; Tumor microenvironment.

Fig. 1

Methylation patterns in MDS/AML and mechanisms of action of AZA and DAC. 1) In normal human cells, CpG islands in the promoter region of tumor suppressor genes are unmethylated (indicated by green dots), allowing transcription of these genes. 2) Hypermethylation of tumor suppressor genes (indicated as red dots) in the pathogenesis of MDS leads to silencing of tumor suppressor genes and development of a leukemic phenotype. 3) Treatment with AZA nucleosides causes demethylation of the hypermethylated CpG islands in MDS/AML leading to reactivation of tumor suppressor genes and anti-leukemic effects

Fig. 2

Structure of azanucleosides. Structure of deoxycitidine and the two azanucleosides azacitidine (AZA) and decitabine (DAC). DAC is the 2′didesoxy form of AZA, incorporated into DNA upon triphosphorylation. AZA is primarily incorporated into RNA. Upon triphosphorylation and reduction by the enzyme ribonucleotide reductase it is also incorporated into DNA. The red circles highlight structural differences between deoxycytidine and the two azanucleosides AZA and DAC. The purple circle highlights the structural difference between AZA and DAC

Fig. 3

Proposed mechanism of HMA-induced IFN response. The figure shows an epithelial tumor cell where the ERV promoters are methylated. Therapy with AZA/DAC leads to demethylation of ERV promoters (1), resulting in transcription of ERV genes, ssRNA and dsRNA (2). In the cytoplasm, ERV dsRNA is sensed by the pathogen recognition receptor (PRR) RIG1 and MDA5, which activate the transcription factors NFκB and IRF3 after binding to the adapter protein MAVS (3). The endosomal membrane-bound TLR-7 and -8 recognize endosomal ssRNA, and activate the transcription factors NFκB and IRF3 after binding to the adapter molecule MyD88 (4). The endosomal membrane-bound TLR-3 recognizes endosomal dsRNA, and activates the transcription factors IRF-5 and -7 after binding to the adapter molecule TRIF (5). These three pathways all drive the expression and secretion of interferon type 1 and 3 (INFI/III) (6). IFNI and III signal back via an autocrine feedback loop and the INF-receptor (IFNR), which signals via JAK/STAT (7). This results in the up-regulation and secretion of the chemokines CXCL9 and 10, which attract tumor-specific CTLs (8). In addition, AIM and ISGs are upregulated, which also aid in reactivation of dormant anti-tumor immunity (9). Furthermore, TAAs are upregulated (10), as are MHC-I molecules (11), which together enhance the immunologic visibility of the tumor cells and enable them to be recognized by the TCR of tumor-specific CTLs. Treatment with HMAs also results in the unwanted up-regulation of inhibitory immune checkpoint receptors (PD-1, CTLA-4) (12) and their ligands (PD-L1, PD-L2, CD80, CD86) (13), which can result in secondary resistance to HMAs, but may also be exploited as a sensitizing or priming strategy for targeted treatment with immune checkpoint modulators

Fig. 4

Taxonomy of retrotransposons. The so-called retrotransposons or class I transposons as opposed to class II (DNA) transposons (not depicted) can be grouped into long terminal repeat (LTR) containing and non-LTR transposons. The best investigated LTR retrotransposons are the human endogenous retroviral elements (ERV). Together with the non-LTR retrotransposons LINE (long interspersed nuclear elements), human ERVs are capable of retrotransposition in an autonomous manner. In contrast, short interspersed nuclear elements (SINEs) like ALU or MIR (mammalian-wide interspersed repeats) sequences cannot perform autonomous retrotransposition. Nevertheless, ALU sequences may be able to move with the help of active LINE elements