Hemoglobin F Induction Is Frequent during Low-Dose 5-aza-2′-Deoxycytidine Treatment of Older AML/MDS Patients.

Introduction: DNA hypermethylation is a frequent epigenetic mechanism for gene silencing in various malignancies. Clinical trials of low-dose DNA methyltransferase (DNMT) inhibitors, e.g. 5-aza-2′-deoxycytidine (Dacogen®, DAC) in AML and advanced MDS have shown encouraging activity, and both global and gene-specific in vivo demethylation (e. g. of the p15/INK4b tumor supressor gene) have been reported and valid surrogate markers for effective epigenetic therapy are warranted. DNMT inhibitors were also successfully used to upregulate HbF in patients (pts) suffering from hemoglobinopathies. We wished to investigate whether HbF induction also occurs during AML and MDS treatment with low-dose DAC. Using K562 and HEL myeloid leukemia cell lines which can be induced to erythroid differentiation by hemin and to megakaryocytic differentiation by phorbol myristate acetate (PMA), we also analyzed in vitro changes in globin transcription induced by DAC.

Results: HbF levels were measured by HPLC in peripheral blood from 36 pts (25 with AML and 11 with MDS, median age 76 years, range 67–84) treated with the identical low-dose DAC schedule (135 mg/m² i.v. over 72 hours). 10 pts had received hydroxyurea (HU) prior to DAC to control leukocytosis. HbF baseline levels varied from 0.1–9.6% (median 0.6%). 22/36 (61%) including 8 pts with prior HU exposed normal HbF levels (≤1.0%), in 14 pts (39%, 2 pts with prior HU) HbF levels were already increased. 27 pts received two or more courses of DAC. In 20/27 pts (74%), an increase in HbF expression during at least one course of treatment could be detected. In 13/15 pts (87%) with initially normal HbF a median increment of 0.8% (0.1–3.7%) was observed, and 7/12 (58%) with initially elevated HbF levels showed further increase (median increment of 1.8%, range 0.7–3.7%) during treatment. Median number of courses until maximum increment was 2 (1–6). In 6 pts HbF levels decreased, in 1 pt HbF expression was unaltered upon DAC treatment.

To investigate mechanisms of this induction, K562 and HEL cells were treated with DAC at non-cytotoxic concentrations, i. e. 100 and 20 nM DAC for 3×24 hrs, respectively. Cells showed striking morphological changes, and by benzidine staining became positive for hemoglobin synthesis. mRNA microarray analyses (HG-U133B gene chip, Affymetrix) revealed significant, strong induction of transcripts for alpha 1-, alpha 2-, A gamma-, epsilon-globin expression in both cell lines, zeta-globin expression was substantially increased in K562 cells, whereas beta- and delta-globin were only expressed at low levels. With PMA treatment only delta-globin expression was increased in both cell lines but other globin transcripts were decreased or unchanged.

Conclusions: HbF is upregulated in the majority of AML/MDS patients upon treatment with DAC, and alpha- and gamma-globin gene induction by DAC can be modeled in vitro. DNA methylation analyses will be necessary to show whether HbF upregulation in AML/MDS patients treated with DAC is associated with demethylation of the gamma-globin locus. Measurement of HbF could serve as rapid and reproducible method for in vivo monitoring of demethylating therapy.