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Page 128 Saliba et al. Cancer Drug Resist 2021;4:125-42 I http://dx.doi.org/10.20517/cdr.2020.95
[38]
One of the ways HMAs exhibit their action is through epigenetic mechanisms . The role of DNA
methylation, specifically cytosine methylation, in the control of gene expression and epigenetic regulation
was identified in different eukaryotic cells in the 1970s [39-41] . It was consequently thought that compounds
capable of changing the patterns of cytosine methylation might impact cell differentiation and differential
gene expression in various cell types that harbor the same genetic information. Azacitidine and decitabine
were subsequently shown to alter differentiation in cultured mouse embryo cells [42-44] . Therefore, the focus
with the use of HMAs shifted from their cytostatic and cytotoxic effects seen at higher doses to their role as
[45]
potent inhibitors of DNA methylation at lower doses and with longer exposure . Importantly, at these low
[46]
doses, HMAs reportedly do not cause cell cycle arrest .

Azacitidine and decitabine are prodrugs that require phosphorylation to monophosphate forms by
uridine-cytidine kinase (UCK) and deoxycytidine kinase (DCK), respectively [38,47] . They are then further
phosphorylated to diphosphate and triphosphate derivatives (5-aza-CTP and 5-aza-dCTP) by pyrimidine
monophosphate and diphosphate kinases. These cytosine derivatives, like their natural counterparts, are
also subject to degradation by cytidine deaminase (CDA).

The fates of 5-aza-CTP and 5-aza-dCTP are somewhat different. Approximately 80%-90% of azacitidine
incorporates into RNA as 5-aza-CTP, resulting in alteration of RNA methylation and inhibition of protein
[48]
synthesis . The RNA-dependent effects of azacitidine are independent of the cell cycle and affect both
messenger and transfer RNA [49-51] . The remaining 10%-20% of azacitidine is converted to 5-aza-dCTP by
ribonucleotide reductase and, like the 5-aza-dCTP that is the main metabolite of decitabine, is incorporated
into DNA.

Once 5-aza-cytosine (5-aza-C) is incorporated into DNA, the downstream effects are the same whether
the altered base is derived from azacitidine or decitabine. When DNMTs act on normal cytosine, they
form a transient covalent bond between a cysteine at the enzyme active site and C6 of cytosine. When
these same enzymes act on 5-aza-C, the same covalent adduct is formed, but it is more stable and cannot
be enzymatically reversed. As a result, cells are left without the enzymatic activity of the adducted DNMT
enzyme molecule and with a new covalent DNA-protein crosslink for each DNMT molecule inhibited [52-59] .
Thus, after HMA treatment, formation of DNA-protein cross-links and loss of DNMT activity are
inextricably linked.

According to current understanding, HMA-induced hypomethylation of promoters of tumor suppressor
genes leads to the interruption of feedback loops between DNA methylation and histone methylation. This
reverses the silenced chromatin state of the histones at the tumor suppressor genes, thus facilitating their
expression, the resulting suppression of leukemogenesis, and the induction of cellular differentiation [45,60,61] .

HMAs also trigger DNA damage response pathways. At high HMA concentrations, phosphorylation of
[62]
histone H2A.x is observed , indicating that one or more of the DNA damage-activated kinases ATM,
ATR or DNA-PK have been activated. The irreversibly bound DNMT can also lead to cell cycle arrest at
[63]
the transition from S to G2 through the activation of the ATM and ATR pathways . The events that occur
downstream of cell cycle arrest and lead to removal of the DNA-DNMT protein cross-links are still being
[64]
elucidated . Current understanding, enhanced by the recent demonstration of DNMT inhibitor/PARP
[62]
inhibitor synergy , suggests that DNMT-DNA covalent complexes might be removed by base excision
repair, although the glycolyase involved and how it is activated remain to be determined. Inhibition of
[67]
DNMT1 may also indirectly affect DNA repair mechanisms [65,66] . Vispé et al. showed that combining
treatment with decitabine and DNMT1 siRNA uncouples DNA damage from DNA demethylation and
again suggests a dual mechanism of action for HMAs.