We have identified and characterized p53R2 as a novel decitabine gene target in human cancer. p53R2 activation links decitabine activity to p53-mediated DNA damage responses, as its induction is p53-dependent. This distinguishes p53R2 from p21/CDKN1A, which we have previously shown is induced by decitabine treatment by both p53-dependent and p53-independent mechanisms (15
). p53R2 induction may thus be an appropriate pharmacodynamic marker for p53 activation by decitabine in vivo
. In MDS, p53 mutations are infrequent, ranging from 6% to 14% in adult MDS patients (17
). Thus, activation of p53R2 and other p53 target genes may be important in mediating therapeutic responses in this setting, consistent with the data presented here. This hypothesis is also supported by our finding that p53R2 contributes to growth arrest in vitro
in tumor cells responding to decitabine treatment. Given the complex nature of cellular gene expression changes following decitabine treatment (34
), it is remarkable that specific knockdown of p53R2 significantly alters decitabine-mediated growth arrest, and suggests that this protein may play a key role in decitabine responses in tumors in which wild-type p53 is retained.
Decitabine treatment induces p53R2 expression in vivo
and its induction correlates with clinical response in MDS/AML. It is unclear whether p53R2 directly contributes to the therapeutic effect of decitabine, or alternatively whether its induction is a surrogate marker for p53 activation, which may contribute to the therapeutic response by a diverse set of mechanisms. Our observation that p53R2
mRNA but not p53R2 protein induction correlates with clinical response seems to support the latter model; however, analysis of a greater number of patient samples is required to adequately address this question. If p53R2 does contribute to therapeutic responses, we hypothesize that it may do so by promoting tumor cell differentiation, a long-established effect of DNMT inhibitors (46
). p53R2, by catalyzing dNTP formation required for DNA repair in growth arrested cells, may allow for enhanced cell survival in the face of genomic DNA hypomethylation. This could in turn promote cellular differentiation by allowing the re-expression of epigenetically silenced genes in surviving hypomethylated cells.
p53R2 is not induced by genomic DNA hypomethylation mediated by genetic knockdown of DNMT1; furthermore, the 5′ CpG island of p53R2 does not display methylation changes during its activation by decitabine either in cell lines or in vivo
. Moreover, etoposide treatment induces p53R2, suggesting that p53R2 is not silenced by DNA methylation in these cancer cell lines, even at potentially cryptic regulatory sites. Taken together, these data unlink DNA hypomethylation from p53R2 induction. Instead, we hypothesize that covalent adduct formation between decitabine-incorporated DNA molecules and DNMT enzymes leads to p53R2 induction. Supporting this model, dose response experiments revealed a tight correlation between the loss of soluble DNMT1 from nuclear extracts and the induction of p53R2
following decitabine treatment. In addition, two other DNMT inhibitors that form covalent adducts, 5-azacytidine and zebularine, also induce p53R2
expression while RG108, which does not form covalent adducts, does not. Despite these data, it is possible that p53R2 is silenced by DNA hypermethylation in certain tumors, although this has yet to been reported. More generally, the fact that p53R2 induction in vivo
appears to be independent of promoter DNA hypomethylation emphasizes the role of hypomethylation-independent gene activation in the activity of decitabine in myeloid malignancies. Additional support for the role of hypomethylation-independent effects comes from an earlier study that found that the majority of decitabine-activated genes in MDS/AML do not show induced promoter DNA hypomethylation following drug treatment (47
p53R2 is a homolog of the R2 subunit of ribonucleotide reductase that pairs with the catalytic R1 subunit to form a competent enzyme complex in non-proliferative cells (48
). In addition to its function in nuclear DNA repair, p53R2 has recently been demonstrated to provide dNTPs for mtDNA replication (49
). Interestingly, p53R2
gene mutations were identified in individuals with severe mtDNA depletion in muscle (49
). Based on these data, it has been suggested that the main function of p53R2 may not be to mediate nuclear DNA repair but rather for mtDNA synthesis (48
). Thus it is of interest to determine whether decitabine treatment alters mtDNA function and whether this could impact cellular responses to DNMT inhibitors.
The original reports on p53R2 used overexpression studies to show that p53R2 induced cell cycle arrest via a G2 phase block (26
). In agreement, our overexpression studies indicate that p53R2 induces G2 arrest in RKO cells (data not shown). However, our siRNA knockdown studies revealed that p53R2 primarily contributes to decitabine-mediated growth arrest by mediating a G1 phase block. Consistent with this observation, it was recently shown that p53R2 contributes to G1 arrest in UV irradiated cells by disassociating from a direct interaction with p21 and facilitating the accumulation of nuclear p21 (51
). It is currently unknown whether p53R2/p21 interactions are involved in cellular growth arrest in response to decitabine; however, it is clear from our data that overexpression studies may not accurately predict the functional role of endogenous p53R2 in mediating cell growth arrest.
In summary, we report that p53R2, a ribonucleotide reductase gene linked to p53-dependent DNA repair, is a robust gene target of decitabine therapy in vitro and in vivo. In addition, we show that p53R2 functionally contributes to cellular responses to decitabine and that its induction directly correlates with clinical responses in MDS/AML. These data open up a number of new lines of investigation relevant for understanding the molecular pharmacology of decitabine and other epigenetic therapies as treatments for human cancer.