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Peixoto et al. Cancer Drug Resist 2018;1:219-29 I http://dx.doi.org/10.20517/cdr.2018.17 Page 223
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of CD8 tumor-infiltrating lymphocytes (TILs) are directly linked to clinical outcome in numerous cancers,
such as breast cancer or ovarian cancer [26,27] . It is now described that this infiltrate is regulated by epigenetic
modifications. For example, the HMT enhancer of zeste homolog 2, which mediates the trimethylation of
histone H3 lysine 27 (H3K27me3), and DNMT1 which repressed the production and secretion of CXCL9
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and CXCL10 by Th1 therefore inhibited the recruitment of CD8 T-cells within the tumor in ovarian cancer
[1]
models . Moreover, decitabine was also found to increase CD80 expression on tumor cells, inducing T
[28]
lymphocytes infiltration in mouse EL4 tumor model and tumor rejection .
Increased expression of PD-1 in breast cancers and colorectal carcinoma was related to both decreased DNA
methylation and decreased repressive H3K9me3 and or H3K27me3 histone marks on the PD-1 promoter [29-31] .
However, no changes in epigenetic modifications were observed on the PD-L1 promoter in these cancers,
probably due to its unmethylated status in normal tissues. Similarly, the expression of the immune
checkpoint inhibitors CTLA-4, TIM-3 and LAG-3 were also up-regulated in breast cancers compared to
normal adjacent tissues and their promoters presented lower DNA methylation and lower repressive histone
[30]
marks . Only one exception was described since the decreased expression of the immune checkpoint
inhibitor LAG-3 appeared to be specifically associated to variations of histone modifications (H3K9me3 and
H3K27me3) [29,32] . Similar results showed an inverse correlation between promoter DNA methylation of PD-
1, PD-L1, CTLA4 and expression of these proteins in NSCLCs compared to normal tissues [33,34] . Moreover,
methylation of the CTLA4 promoter was also inversely correlated to its expression in a large cohort (470) of
malignant melanoma patients treated with anti-PD-1/PD-L1 antibodies. Furthermore, a strong significant
correlation was observed between low promoter methylation of CTLA4 and both positive response to
[35]
immunotherapy and survival . In prostate cancers, methylation of PD-1 promoter was more frequently
[36]
observed than in normal tissues and its expression was inversely correlated to its promoter methylation .
Moreover, patients presenting a high level of methylated PD-1 promoter presented a higher risk of
recurrence in prostate cancer. In leukemia, PD-L1 expression has also been associated with low promoter
[37]
methylation and to poor prognosis . The demethylation of an enhancer region of PD-1 and the protein re-
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[38]
expression was reported in CD8 T-cells in chronic lymphocytic leukemia . In diffuse low grade glioma
(LGG), methylation of PD-L1, PD-L2 and CTLA-4 promoters was inversely correlated to their expression.
We also recently reported in epithelial to mesenchymal transition-induced lung cancer cell models (A549)
that transforming growth factor β (TGFβ), a cytokine frequently expressed in the tumor microenvironment,
was responsible for the demethylation of the PD-L1 promoter by inhibiting both global and local DNA
[39]
methylation . In addition, the combined treatment of these cells with TNFα favored NFκB signaling and
PD-L1 transcription by inducing the recruitment of NFκB/p65 on the PD-L1 promoter. Besides the tumor
microenvironment which could severely affect PD-1/PD-L1 expression, cancer drugs might also modify their
expression, thus inducing drug resistance. For example, 30% of melanoma patients treated with ICB who
presented a positive response towards the protocol, developed secondary tumors (for a review ). Exhausted
[40]
T-cells which appeared during ICB are supposed to present an epigenetic-linked genome wide regulation
mediated by the de novo DNMT3A leading to the inhibition of proliferation and metabolic activity and
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thus restricting clonal CD8 T-cell diversity. Indeed, DNA methylation inhibitors improved T-cell response
[41]
in anti-PD-1 therapy . On the opposite, an acquired resistance was frequently found in myelodysplastic
syndrome (MDS) in which the use of DNA hypomethylating agents is the standard treatment. Inhibition
[42]
of DNA methylation induces an increase in PD-1 in T-cells of these patients . Although mechanisms of
resistance to demethylating agents are still unclear, it has been suggested that the activation of the immune
checkpoint inhibitor was more likely involved. Moreover, treatment of NSCLC patients with conventional
chemotherapy or epidermal growth factor receptor inhibitors increased PD-L1 expression unlike anti-PD-L1
[34]
treatments which significantly decreased PD-L1 expression . In addition, an anti-PD-L1 treatment of
primary tumors which expressed low levels of PD-L1, induced secondary tumours presenting an increase in
the methylation of PD-L1 promoter.