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Disease relapse is a major cause of treatment failure following alloSCT for acute myeloid leukemia (AML). Azacitidine has shown efficacy in treating, and preventing, post-transplant relapse in patients with AML. Post-SCT azacitidine administration is challenging due to the possibility of myelosuppression and an incomplete understanding of the optimal dose and schedule. DNMT1 is responsible for genome methylation in S phase and degraded after bonding irreversibly to substituted DNA. Hence, the DNMT1 level may be an attractive pharmacodynamic (PD) endpoint for azacitidine therapy.
We developed a novel DNMT1 assay by flow cytometry as a PD endpoint for hypomethylating agent therapy. To validate the assay, HCT116-wild type (WT) and DNMT1-double-knockout (DKO) cells were incubated with 0.5 µM decitabine, and were harvested, fixed, and processed for flow cytometry using DNMT1 antibody in an indirect assay and co-stained with DAPI to measure DNA content. We assessed the time-dependent depletion of DNMT1 with decitabine incubation (Fig. A).
We subsequently assessed DNMT1 in fixed peripheral blood leukocytes using the same method in 4 AML patients treated with post-alloSCT azacitidine; either parenterally for post-SCT relapse (n=2) or after oral azacitidine maintenance on clinical protocol NCT 01835587 (n=2). Peripheral blood samples were drawn before and 2-6 weeks after starting azacitidine. Immature myeloblasts were identified by co-staining for CD34 and CD117.
We demonstrated depletion in DNMT1 in S-phase CD34+CD117+ cells after azacitidine in the 4 studied paired samples. The degree of depletion was more pronounced with parenteral versus oral azacitidine. In one patient who responded favorably to azacitidine, depletion of DNMT1 correlated with reduction in peripheral blood blasts (CD34+117+ cells) as shown below (Fig. B).
These preliminary data suggest that the DNMT1 assay is a robust single cell approach to assess PD of hypomethylating agents. After further validation, this DNMT1 assay may be a valuable tool to optimize the dose and schedule, and hence the safety and efficacy of post-alloSCT hypomethylating therapies.
Figure A: Depletion of DNMT1 in HCT116-WT and DKO cells cultured with 0.5μM decitabine. B: Depletion of DNMT1 in immature myeloid cells (CD34+CD117+) by flow cytometry in a patient with relapsed AML post-transplant who was treated with parenteral azactidine. The pre-treatment peripheral blood sample was collected at T+32 after reduced-intensity allo-HCT and before starting parenteral azacitidine (40 mg/m2 IV daily for 5 days). The post-treatment sample was collected 45 days after start of azacitidine. The DNMT1 level was determined in CD34+CD117+ and S-phase cells by flow cytometry.