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Franca et al. Cancer Drug Resist 2019;2:256-70 I http://dx.doi.org/10.20517/cdr.2019.004 Page 265
[77]
to those without this allele . The same SNPs, together with RAC1 SNP rs34932801, was genotyped in
59 IBD pediatric patients and no association was found with thiopurine effectiveness after 12 months of
[78]
therapy . In contrast, a retrospective report on a cohort of 156 thiopurine-treated CD adults has shown an
association between the GC heterozygous genotype in SNP rs34932801 and poorer response to thiopurines,
[79]
in comparison to wild-type GG .

Epigenetics
Epigenetic factors, in particular considering molecular factors that can be inherited from one generation
to the other but that do not depend on the DNA sequence, such as methylation of cytosines on DNA and
non-coding RNA profiles, such as microRNAs (miRNAs) and long non-coding RNAs, have been recently
considered as innovative pharmacogenomic determinants of interindividual variability in drug efficacy and
[80]
toxicity .
For thiopurines, studies on the effect of pharmacoepigenetic factors are still limited. Thiopurines have been
[81]
shown to reduce global DNA methylation . This effect was demonstrated in vitro in human leukemia
cells and has been related to the reduction of de novo purine synthesis (DNPS) induced by thionucleotides,
which determines reduction in ATP levels and depletion of S-adenosylmethionine (SAM) concentration,
an important co-factor of methyltransferases, including DNA methyltransferases, enzymes involved in the
[82]
regulation of DNA methylation . Also TPMT activity, by modulating the capability of thionucleotides to
inhibit DNPS, has been shown to influence the reduction of DNA methylation induced by mercaptopurine
[83]
and thioguanine . However, how this effect of thiopurines on DNA methylation may be related to
interindividual variability in efficacy and adverse effects of these drugs or on the expression of candidate
genes involved in thiopurines pharmacokinetics and pharmacodynamics is still unknown.

In the treatment of pediatric ALL, mercaptopurine is generally associated with methotrexate, both during
consolidation and maintenance therapies. The epigenetic factors influencing interindividual variability in
response to these phases of therapy may be of relevance also for thiopurine effects. For examples, a major
adverse effect of consolidation therapy for pediatric ALL is oral mucositis, which is generally ascribed to
methotrexate. However, as already mentioned, in some protocols, such as SJCRH Total XIIIB protocol,
[70]
this adverse effect has been related also to TPMT activity , which indicated that also interindividual
variability in mercaptopurine disposition may influence this adverse effect. A recent study evaluated DNA
methylation profiles associated with oral mucositis during consolidation therapy in children with ALL. This
study confirmed also in patients a significant reduction in SAM levels during methotrexate/mercaptopurine
therapy (doses respectively 5 g/m every 2 weeks with leucovorin rescue 15 mg/m /dose and daily
2
2
2
mercaptopurine 25 mg/m ). In this study, DNA methylation was measured considering the percentage of 12
[84]
CpG islands in the LINE1 gene, that is considered a surrogate for DNA global methylation . Intriguingly,
a small increase in this DNA methylation marker was observed during therapy, with no correlation with
SAM levels and incidence of oral mucositis. One limitation of this study is that DNA was extracted from
peripheral blood mononuclear cells and since DNA methylation is cell specific, different effects could
be observed in tissues more directly involved in methotrexate/mercaptopurine, effects such as residual
lymphoblasts and epithelial cells of the gastrointestinal tract.

Considering non coding RNAs, studies are also limited, in particular for the association with the clinical
effects of thiopurines. One study considered miRNA profiles after treatment with mercaptopurine in rat
placenta, as a model to study toxicity of this drug in fetal development. Differentially expressed miRNAs
[85]
could be identified and were involved in process such as apoptosis, inflammation and ischemia .

Non coding RNAs affecting the expression of pharmacogenes in human liver have been identified and
associated with age. Among these genes, also those relevant for thiopurines pharmacokinetics have been
considered . Results showed that some miRNAs were associated with both age and TPMT expression; in
[86]