A wide range of cancers are notorious for exploiting an immune checkpoint protein called PD-L1 in order to suppress the immune attack. Drugs that inhibit PD-L1 binding has achieved such a remarkable clinical improvement that the word “cure” can now be used for a subset of patients with metastatic melanoma. However, a concerning limitation is that the majority of patients do not respond to this treatment. One of the greatest barrier to overcoming this problem is the lack of understanding of the underlying mechanisms which regulates PD-L1 expression. We aimed to investigate the role of DNA methylation (a fundamental epigenetic mechanism) in regulating PD-L1 expression.

1. DNA methylation regulates PD-L1 expression by activating
endogenous retrovirus elements and the viral mimicry pathway
Ahn, A.1, Rodger, E.J.1,2, Stockwell P.A.1, Parry M.,3 Motwani J.1, Gallagher S.J.4, Shklovskaya E.4, Tiffen J.4, Hersey P.4, Chatterjee, A.1,2 † Eccles, M.R.1,2 †
Background
A wide range of cancers are notorious for exploiting an
immune checkpoint protein called PD-L1 in order to suppress
the immune attack (1). Drugs that inhibit PD-L1 binding has
achieved such a remarkable clinical improvement that the
word “cure” can now be used for a subset of patients with
metastatic melanoma.
However a concerning limitation is that the majority of patients
do not respond to this treatment (2). One of the greatest
barrier to overcoming this problem is the lack of understanding
of the underlying mechanisms which regulates PD-L1
expression.
Melanoma cells that constitutively express PD-L1 (PD-
L1CON) have reduced levels of genome-wide
hypomethylation. This was largely derived from
intergenic regions.
The expression of 9 ERV elements were
determined in 6 PD-L1 low expressing (PD-
L1IND) and 6 PD-L1 high expressing (PD-
L1CON) cell lines using RT-qPCR. 5 out of 9
ERV genes were significantly upregulated in
the PD-L1CON group whereas the remaining 4
did not shown any differential expression (not
shown here).
Methods
Summary
References
1 - Sun C, Mezzadra R, Schumacher TN. Regulation and Function of the PD-L1 Checkpoint. Immunity. 2018;48(3):434-52.
2. - Zerdes I, Matikas A, Bergh J, Rassidakis GZ, Foukakis T. Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer:
biology and clinical correlations. Oncogene. 2018.
1Department of Pathology, University of Otago, Dunedin, NZ. 2Maurice Wilkins Centre, NZ. 3Department of
Mathematics & Statistics, University of Otago. 4The Centenary Institute, University of Sydney
PD-L1 expression is associated with global hypomethylation
which is mostly derived from intergenic regions
Expression of ERV elements are increased in PD-L1 cell lines
(A) Expression of genes involved in the viral-mimicry pathway was increased in the PD-L1 positive cell lines
(samples clustered on the right) compared to the PD-L1 negative cell lines (samples clustered on the left). (B)
Upregulation of the viral-mimicry pathway was also observed after induction of global hypomethylation using
DNMT inhibitor drug treatment. (C) The association of PD-L1 expression with the activation of the viral-mimicry
pathway, was validated in patient tumors using TCGA RNA-seq data. Patients were separated into low PD-L1
expression (group 1, blue) and high PD-L1 expression (group 2, red) groups. Patient tumors with high PD-L1
expression showed an increased expression of genes involved in the viral-mimicry pathway.
Contact details
We aimed to investigate the role of DNA methylation (a
fundamental epigenetic mechanism) in regulating PD-L1
expression.
Aim
Future work
• Confirm the role of the viral mimicry pathway or other pathways in activating PD-L1 levels by using siRNA
knockdown experiments.
• Evaluate the increased expression of all annotated ERV genes in PD-L1 expressing cell lines using RNA-seq
data.
• Investigate the role of non-coding RNAs in regulating PD-L1 expression.
• Melanoma cells that constitutively expressing PD-L1 (PD-
L1con) have reduced methylation levels in intergenic
regions which contain Endogenous Retrovirus (ERV)
elements.
• PD-L1 expression is associated with an increased mRNA
expression of ERV elements.
• PD-L1 expression is associated with an activated viral
mimicry pathway.
Genes involved in the viral mimicry pathway are upregulated
in 1) PD-L1 expressing cell lines, 2) upon DNMTi treatment
and 3) in patient tumors with higher PD-L1 expression
PD-L1 expression is associated with hypo-methylation of ERV elements which reside in intergenic regions. This
upregulates the expression of ERV genes which then stimulates the innate immune response signaling pathway.
This pathway is also referred to as ”viral-mimicry” pathway because the cells are ”tricked” into behaving as if it was
infected with a virus. Overall, this supports the usage of combining epigenetic therapy with immunotherapy.
Conclusion
Endogenous Retrovirus (ERV) elements were
hypomethylated in the PD-L1 high expressing (PD-
L1CON) cell lines compared to PD-L1 low expressing
cell lines (PD-L1IND)
A B C
PD-L1 is characterised with:
methylation of ERV
elements
expression of ERV
elements
activation of the viral
mimicry pathway
PD-L1IND
Melanoma cell lines with inducible
or low PD-L1 expression
PD-L1CON
Melanoma cell lines with constitutive
or high PD-L1 expression
Methylation
Methylation
11 Cell lines
8 BRAF mutants
2 NRAS mutant
1 BRAF and NRAS wildtype
10 Cell lines
5 BRAF mutants
3 NRAS mutant (Q61K)
2 BRAF and NRAS wildtype
† Joint senior authorship
Antonio.ahn@otago.ac.nz Aniruddha.chatterjee@otago.ac.nz
Euan.Rodger@otago.ac.nz Michael.eccles@otago.ac.nz