DNA Hypomethylation Is Not Sufficient to Account for Decitabine Mediated Cytotoxicity in TP53-Mutated Myeloid Malignancies

TP53-mutated myeloid malignancies (TP53-MN) are associated with primary resistance to standard induction chemotherapy and a dismal prognosis, with a median survival of 6-9 months. Treatment with a hypomethylating agent (decitabine or azacitidine) is the standard of care for TP53-MNs, but responses are usually transient and durable remissions uncommon. We previously reported that TP53-MN are selectively sensitive to the combination of decitabine and ATR inhibition. Indeed, synergistic killing to TP53-mutated AML cell lines and primary TP53-MN was observed both in vitro and in vivo. A clinical trial based on these observations is in development. Here, we report our efforts to characterize molecular mechanisms by which loss of TP53 sensitizes cells to decitabine-induced DNA replicative stress and ATR inhibition.

Decitabine is a nucleoside analog that incorporates into DNA and mimics a hemi-methylated CpG, which is recognized by DNMT1, the major maintenance DNA methyltransferase. DNMT1 forms a covalent linkage to this abnormal base, resulting in DNMT1 degradation and subsequent global hypomethylation. We show that decitabine treatment of AML cell lines induces DNA replicative stress as assessed by DNA fiber assays and activation of the ATR/p53 pathway. Resolution of replicative stress is significantly impaired in isogenic lines lacking TP53, resulting in the accumulation of single-strand, and eventually double-strand, DNA breaks. Consistent with an ATR-dependent activation of the G2/M mitotic checkpoint, treatment with decitabine resulted in the accumulation of cells in G2/M, which was significantly accentuated in TP53-deficient cells. CDKN1A (p21) is a p53 target gene that activates the G1/S mitotic checkpoint and helps regulate DNA replication fork speed. To determine the contribution of p21 to decitabine sensitivity, we generated multiple independent CDKN1A^-/- MOLM13 cell lines. Treatment with decitabine induced a similar G2/M block in TP53-deficient or CDKN1A^-/--deficient cells. Moreover, CDKN1A^-/--deficient cells were more sensitive to decitabine alone than wildtype cells (IC50: 110 vs 277 nM, respectively). These results suggest that loss of p21 activity in TP53-MN significantly contributes to their sensitivity to decitabine.

Induction of global DNA hypomethylation is widely considered to be a major mechanism by which decitabine (and azacitidine) induce therapeutic responses in myeloid malignancies. To test this hypothesis, we analyzed the impact of a reversible DNMT1-specific inhibitor (DNMT1i) in isogenic wildtype or TP53-deficient MOLM13 cells. This inhibitor blocks the enzymatic activity of DNMT1, inducing global DNA hypomethylation, but it should not generate covalent DNA-protein adducts. We show using whole genome bisulfite sequencing that treatment with DNMT1i induces a deep, global DNA hypomethylation that is similar to that seen with decitabine. Inhibition of DNMT1 was sufficient to induce DNA replicative stress in TP53-deficient cells. However, it did not induce measurable ATR activation, as assessed by CHK1 phosphorylation. Moreover, whereas decitabine induced double-strand DNA breaks and gH2AX activation in TP53-deficient cells, this was not observed with DNMT1 inhibition. Finally, we assess the ability of DNMT1 inhibition to induce synergistic killing in combination with ATRi of TP53-deficient MOLM3 cells. In sharp contrast to decitabine plus ATRi, which induced strong synergistic killing of TP53-deficient MOLM3 cells (ZIP synergy score = 10.1), no synergy was seen with DNMT1i treatment (ZIP score = 2.97). Collectively, these data suggest that global DNA hypomethylation, while contributing to DNA replicative stress, is not sufficient to mediate the full cytotoxic effects of decitabine in TP53-mutated myeloid malignancies.

No relevant conflicts of interest to declare.