5-Aza-2′-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair

Abstract

5-Aza-2′-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.

Evaluation of noncytotoxic DNMT1-depleting therapy in patients with myelodysplastic syndromes

“…(B) The sugar in decitabine is natural, enabling DNA incorporation without terminating chain elongation. This shifts the DNA damage curve to the right compared with cytarabine (10,14,33,34), even though the decitabine-DNMT1 DNA-protein cross-link requires repair by homologous recombination (35)(36)(37). The DNA repair delays cell-cycle progression; however, concentrations can be found that deplete DNMT1 without cytotoxicity (green zone), the intent in this clinical trial (unlike with cytarabine, a lower dose need not imply lower efficacy).…”

“…The pyrimidine ring modification of decitabine subsequently depletes DNMT1 and can also directly hypomethylate DNA, since it cannot accept a methyl group (33,34). High decitabine concentrations, however, do have antimetabolic, DNA-damaging cytotoxic effects (35)(36)(37). Moreover, aggressive DNA-damaging cytotoxic therapy, despite the risk of treatment-related mortality, cures some myeloid cancers (e.g., AML subtypes that do not have p53 system abnormalities); this suggested that cytotoxicity could be the key to durable remissions.…”

Demethylation Therapy as a Targeted Treatment for Human Papillomavirus–Associated Head and Neck Cancer

“…S2) that was shown to activate p53 in HPV þ cells (30). However, DNA damage, induced by increasing concentrations of 5-aza (27), can further contribute to p53 stabilization. We recently reported that HPV is not integrated in a significant portion of HPV þ HNSCC suggesting that HPV episomal maintenance is required in these tumors (8,14).…”

“…2A). High concentrations of demethylating drugs were shown to induce DNA damage in cells (27). To test whether DNA damage after 5-aza treatment contributed to 5-aza-induced p53 upregulation, we treated UMSCC47 cells with radiomimetic drug zeocin, or 1 or 3 mmol/L of 5-aza-the concentrations that significantly downregulated HPV E6 expression.…”

“…Furthermore, 5-aza is incorporated into RNA (24,25), where it can alter mRNA stability (26) and therefore change expression independently of its effect on transcription. Finally, 5-aza and decitabine cause DNA damage and activate the DNA damage response in some cells; however, the nature of this DNA damage has not been fully characterized and is likely cell type-dependent (27).…”