The

n e w e ng l a n d j o u r na l

of

m e dic i n e

Cl inic a l I m pl ic a t ions of B a sic R e se a rch

ElizabethG. Phimister, Ph.D., Editor

Epigenetic Modulators and the New Immunotherapies

AnthonyE. Dear, M.B., B.S., Ph.D.
The recent arrival of the immune-checkpoint
inhibitor drugs first applied to melanoma
and more recently tested as a treatment for different solid tumors and hematologic cancers
has changed the management of these conditions. The response rates have been impressive,
as have the improvements in overall survival over
the course, in some instances, of several years.
However, the lack of response to single-agent
treatment and the emergence of resistance have
been problematic.1 Combinatorial therapeutic
strategies have been used with varying degrees
of clinical success.2
New experimental approaches, such as that
recently described by Chiappinelli et al.3 and
Roulois et al.,4 are therefore welcome. These investigators identified an immune-based mechanism of action of the epigenetic modulator class
of agents called the DNA methyltransferase inhibitors, which are currently used in the treatment of hematologic cancers. This work points
to a combination of new immunotherapeutic
agents of the immune-checkpointinhibitor
class and epigenetic modulators.
Chiappinelli and colleagues (in Li et al.5) had
previously reported that treatment of cancer
cells with a DNA methyltransferase inhibitor,
azacytidine, up-regulates the expression of genes
in immunoregulatory pathways, especially those
that regulate interferon signaling. In their more
recent study, they identified a curious mechanism that underlies the azacytidine-mediated
immune response and showed that exposure to
azacytidine sensitizes melanoma cells to immune-checkpointinhibitor therapy in a preclinical melanoma model.3
The mechanism described by Chiappinelli et
al. involves the detection of cytosolic doublestranded RNA (dsRNA) through the RNA-sensing proteins TLR3, MDA5, and RIG1. The expression of these proteins in several cell types is
684

up-regulated in response to viral infection and

triggers the interferon type I response.3 The authors hypothesized that azacytidine-mediated
activation (perhaps more accurately described as
derepression) of endogenous retroviral sequences, which are usually epigenetically silenced by
gene-promoter DNA methylation, triggers the
dsRNA-sensing pathway and downstream interferon type I response (Fig.1). The azacytidinemediated induction of several endogenous retroviral sequences was found to be correlated
temporally with the expression of viral-defense
genes (and not proteins), which supports this
hypothesis3 (Fig.1). In vivo correlative geneexpression studies of primary endothelial ovarian
cancers subsequently showed that high levels of
expression of endogenous retroviral sequences
in tumors track with high levels of expression of
viral-defense genes.3
The authors then evaluated levels of azacytidine-mediated induction of viral-defense gene
expression in melanoma and ovarian, breast,
colon, and lung cancers from the Cancer Genome Atlas studies. They sorted the tumors into
low-expressing and high-expressing groups and
showed that tumors with high levels of expression of the viral-defense set of genes were more
likely to be associated with a comparatively good
clinical outcome, which prompted a question: Is
the activation of the viral-defense pathway associated with the response to immune-checkpoint therapy? The answer would seem to be yes.
The authors found a high level of expression of
viral-defense genes in the tumors of patients
who received long-term benefit from anticytotoxic T-lymphocyteassociated protein 4 (CTLA4) therapy, and they found a low level of expression in the tumors of those who did not have
long-term benefit from this therapy. They did
not report a parallel analysis involving patients
who did not receive antiCTLA-4 therapy.3

n engl j med 374;7nejm.org February 18, 2016

The New England Journal of Medicine

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Copyright 2016 Massachusetts Medical Society. All rights reserved.

Clinical Implications of Basic Research

A
Tumor cell

C YT O PL AS M

Azacytidine
(a DNMT inhibitor)

IFNAR1
and IFNAR2

CYTOPLASM

No methylation;
derepression of
endogenous retroviral
sequences

dsRNA-sensing proteins
bound to dsRNA

IFNAR1
and IFNAR2
Methylation
M

MDA5

Interferon-
RIG1

Endogenous
retroviral dsRNA

TLR3

N U C LE U S

Induced transcription of
the interferon- gene
IRF3
MAVS

MI T OC HONDRI ON

NF-B

IRF7

DNA

B
Interferonstimulated
genes

NU CLEU S

C Y T OP LASM

Cytokines

STATs
IFNAR1
and IFNAR2

AntiCTLA-4
therapy

Antigen from cancer

cell (e.g., cancer
testis antigen)

IRF9

MHC
class I

Interferon-
IFNGR1

T-cell
receptor
Interferon-

A C T IV A T E D
T C E LL

Figure 1. Enhancing Tumor-Cell Visibility to the Immune System with the Use of Viral Mimicry A Model.
Chiappinelli et al.3 and Roulois et al.4 recently reported that drug-induced derepression of endogenous retroviral sequences (ERVs) sets off a
chain of events that results in the synthesis and secretion of interferon-. The transcribed ERVs are recognized by double-stranded RNA (dsRNA)
sensing proteins (MDA5, RIG1, and TLR3). These proteins either directly or indirectly (through the mitochondrial antiviral signaling protein
[MAVS]) up-regulate the expression of interferon by inducing translocation of interferon regulatory factor (IRF) 3, nuclear factor B (NF-B),
and IRF7 to the nucleus, thereby inducing transcription of IFNB1 (Panel A). Interferon- in turn stimulates interferon / receptors 1 and 2
(IFNAR1 and IFNAR2) and activates IRF9 and signal transducers and activators of transcription (STATs), which ultimately results in a cytokinemediated antivirus response (Panel B). Up-regulation of antiviral cytokines and cross-talk between IFNAR1 and IFNAR2 and the interferon-
receptor (IFNGR1) result in induction of cell-surface expression of major histocompatibility complex (MHC) class I peptides (e.g., cancer testis
antigen) and activation of T-cell receptormediated immunity. Chiappinelli et al. also reported data supporting the hypothesis that drug-induced
derepression of ERVs sensitizes cancer cells to anticytotoxic T-lymphocyteassociated protein 4 (antiCTLA-4) therapy. DNMT denotes DNA
methyltransferase, M methylation, and P phosphorylation.
n engl j med 374;7

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February 18, 2016

685

The New England Journal of Medicine

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Clinical Implications of Basic Research

Finally, the investigators evaluated the effect

of azacytidine treatment on immune-checkpoint
therapy in a mouse model of melanoma. Azacytidine treatment enhanced the responsiveness of
tumors to antiCTLA-4 therapy, and azacytidine
pretreatment of melanoma cells before injection
resulted in complete clearing of melanoma cells
in mice that were given antiCTLA-4 treatment.3
An important line of investigation would be to
close the loop that is, to show that the
therapeutic response is driven by lymphocytes
that specifically bind tumor neoantigens on
tumor cells that have high levels of expression
of viral-defense genes.
Roulois et al.4 also described the presence of
a dsRNA-based sensing system in colon-cancer
cells, with molecular components similar to those
described by Chiappinelli et al.,3 leading to DNA
methyltransferase inhibitormediated up-regulation of endogenous retroviral sequences. They,
too, observed that up-regulation of this dsRNA
sensing system is associated with the up-regulation of the interferon response, which they described as viral mimicry, to which they attributed the antitumor effects of the DNA
methyltransferase inhibitor.4
Taken together, these observations provide a
mechanistic explanation for the epigenetic modulation of the immune response that has been
observed in previous studies, and they point to
the tantalizingpossibility that treatment for
multiple tumor types can be enhanced by com-

bining epigenetic or immune sensitizing therapeutic agents,such as the DNA methyltransferase

inhibitors, with immune-checkpoint inhibitors,
such as those that block CTLA-4 and programmed death 1 (PD-1). Another important
line of investigation will be the testing of viraldefense gene expression signatures in tumors as
a basis for the selection of treatment strategies.
Active early-phase clinical trials of combination
treatment with DNA methyltransferase inhibitors
and checkpoint inhibitors (e.g., ClinicalTrials.gov
number NCT01928576) may prove informative.
Disclosure forms provided by the author are available with the
full text of this article at NEJM.org.
From the Eastern Clinical Research Unit, Translational Research
Division, Department of Medicine, Eastern Health Clinical
School, Monash University, Melbourne, VIC, Australia.
1. Kelderman S, Schumacher TN, Haanen JB. Acquired and in-

trinsic resistance in cancer immunotherapy. Mol Oncol 2014;8:

1132-9.
2. Sathyanarayanan V, Neelapu SS. Cancer immunotherapy:
strategies for personalization and combinatorial approaches.
Mol Oncol 2015 October 23 (Epub ahead of print).
3. Chiappinelli KB, Strissel PL, Desrichard A, et al. Inhibiting
DNA methylation causes an interferon response in cancer via
dsRNA including endogenous retroviruses. Cell 2015;162:97486.
4. Roulois D, Loo Yau H, Singhania R, et al. DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous transcripts. Cell 2015;162:961-73.
5. Li H, Chiappinelli KB, Guzzetta AA, et al. Immune regulation by low doses of the DNA methyltransferase inhibitor
5-azacitidine in common human epithelial cancers. Oncotarget
2014;5:587-98.
DOI: 10.1056/NEJMcibr1514673
Copyright 2016 Massachusetts Medical Society.

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n engl j med 374;7nejm.org February 18, 2016

The New England Journal of Medicine

Downloaded from nejm.org at WEILL CORNELL MEDICAL COLLEGE LIBRARY on March 18, 2016. For personal use only. No other uses without permission.
Copyright 2016 Massachusetts Medical Society. All rights reserved.