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Page 224 Peixoto et al. Cancer Drug Resist 2018;1:219-29 I http://dx.doi.org/10.20517/cdr.2018.17

A recent multi-country (France, Spain and Italy) cohort study of 142 high grade NSCLC samples highlighted
a DNA methylation specific signature which can significantly predict which patients could positively
[43]
benefit from nivolumab or pembrolizumad treatments . DNA repair pathways have been associated with
efficiency of ICB treatments. Indeed, a study analyzing the methylation status of 179 genes involved in DNA
repair in HNSCC, cervical carcinoma and laryngeal squamous cell carcinoma identified 15, 3 and 2 genes,
respectively, whose methylation was associated with increased PD-L1 expression. Amongst these genes,
RAD51B was found in the three different cancer models suggesting that alteration of homologous DNA
[44]
reparation strongly favored immune checkpoint inhibitor expression .

Only 15% of metastatic HNSCC presented an objective response to anti-PD-1/PD-L1 treatments. In these
cancers, both PD-L1 and PD-L2 expression was increased in tumor cells compared to normal adjacent
[45]
tissues and their expression was inversely correlated to methylation of their promoters . Interestingly,
human papillomavirus-positive HNSCC were strongly associated with both PD-1 and PD-L1 promoter
methylation and poor prognosis suggesting that these particular tumors are unlikely to respond to
immunotherapy [45,46] . Treatment of mesothelioma cells using the demethylating agent decitabine alone is
unable to significantly restore PD-L1 expression but combination of decitabine with HDACi [valproic acid,
[30]
suberoylanilide hydroxamic acid (SAHA) or NODH] strongly induced PD-L1 transcription in these cells .
Expressions of PD-L1 and CTLA4 mRNA in acute myeloid leukemia (AML) patients treated with combined
HDACi and DNMTi were also increased during treatment but presented very high oscillations. Interestingly,
decitabine alone was sufficient to restore both mRNA and protein PD-L1, PD-1 and CTLA4 expression in a
[47]
dose-dependent manner in AML cell lines .

COMBINATION OF EPIDRUGS WITH IMMUNOTHERAPIES RESTORES TREATMENT
EFFICIENCY
Cell models
Although some studies showed a positive effect of 5-Aza treatment on PD-L1 expression in NSCLC cell lines,
only 13% of NSCLC tumors issued from patients treated with this demethylating agent, presented a strong
expression of PD-L1 protein (however, PD-L1 expression was at least detectable in 55% of cases). These data
strongly support the hypothesis that different cellular mechanisms control PD-L1 expression. Indeed, the
competitive inhibitor of DNMT1, procainamide or 5-Aza treatments alone failed to induce PD-L1 expression
in the NSLCC cell line A549 whereas the combination of these compounds with TNFα reactivated PD-L1
expression . In NSCLC models (A549 and H838 cells), the DNA methylation inhibitor Aza, induced the
[39]
demethylation of promoters, increased the expression of interferon regulating factor 1 (IRF1) and provoked
an increase in PD-L1 expression in both a interferon-dependent and independent manner . Moreover,
[48]
expression of CXCL9 and CXCL10, consecutive to the IRFs activation, improved the efficiency of anti-PD-L1
protocols in mice models.

Pre-clinical studies
Despite obtaining better therapeutic responses with combined molecules than single drug treatment,
combined anti-PD-1 and anti-CTLA4 therapy could not limit the formation and tumor growth of CT26
[49]
and 4T1 cancer models in mice . However, HDACi significantly increased PD-L1 expression in the
4T1 cell model, and tri-therapies with the further addition of 5-Aza or entinostat fully eradicated the
tumors [49,50] . Indeed, the authors showed that the use of epigenetic drugs led to the elimination of the potent
immunosuppressive myeloid-derived suppressor cells (MDSCs) at very low concentrations, doses generally
well tolerated by cancer cells. Moreover, the addition of antibodies targeting MDSCs, or the use of a PI3K
inhibitor known to reduce circulating MDSCs, in combination with immunotherapy led to the same
efficiency. Similar results were observed in NSCLC cells treated with Mocetinostat (one inhibitor of HDAC I/
IV classes) . On the opposite, HDAC11 knock-out mice presented very high levels of immunosuppressive
-/-
[51]