Apoptosis Induction and Cell Cycle Profiling after Treatment with Low-Dose Decitabine and/or Bortezomib in the AML Cell Line Kasumi-1

Introduction : Acute myeloid leukemia (AML) is a heterogeneous clonal hematopoietic neoplasm characterized by maturation arrest in the myeloid lineage. The standard of care regimen of induction for AML patients is an anthracycline and cytarabine combination, followed by consolidation therapy. Nevertheless, relapse still remains a major hurdle for successful AML chemotherapy and alternative treatment strategies are much needed. Epigenome-modifying drugs, such as decitabine (D), a DNA methyltransferase inhibitor (DMTi), have shown promise as therapeutics for AML in low doses, while higher doses are associated with cellular differentiation and cytotoxicity. On the other hand, oxidative stress inducers have been strongly implicated in the targeting of LSCs, since increased oxidation is associated with reduced self-renewal, which in turn leads to either differentiation or death of haemopoietic cells. Nevertheless, the induction of oxidative stress alone is not sufficient for AML treatment.

Aims: Since only approximately 50% of patients treated with DMTis show a hematological improvement (HI) or better, and few alternative treatments exist for patients who fail to respond, the aim of this study is to investigate the possible reversal of decitabine resistance phenotype in AML, in terms of apoptosis and cell cycle profiling, by the combination of decitabine with oxidative stress inducing agents, such as the proteasome inhibitor bortezomib (BZ).

Methods : The AML Kasumi-1 cells, carrying the t(8;21) and the KIT mutation N822, were cultured in RPMI 1640, supplemented with 20% FBS, and incubated in 5% CO₂ at 37°C. Cells were treated with low-dose decitabine (10nΜ, 50nΜ, 100nM, 200nM and 400nΜ), with or without bortezomib (10nM) for 24h. Flow cytometry was used for apoptosis (Annexin V/PI staining) and cell cycle profiling (DAPI staining). One-way Anova and LSD/ Bonferroni methods were applied for the statistical analysis of the results.

Results : Our data indicate significant alterations in cell death and cell cycle stages in Kasumi-1 cells following D and BZ combination treatment compared to the control (untreated cells) and single treatments. Apoptosis was statistically significantly increased compared to control (15,15%) after only 100nM and 400nM of single D treatment (27,2% and 28,25%, p=0.037 and p=0.026, respectively) and after D/BZ treatment for all D concentrations tested [31,5%, 33,4%, 54,65%, 47,1% and 55,55%, from lower to higher concentration, with p=0.008 and p=0.004 for 10nM and 50nM respectively, and p<0.000 for 100nM, 200nM and 400nM). Compared to single treatments, the D/BZ combination significantly increased apoptosis by 101,92%, 72%, 100,9%, 113,12% and 96,63% (p=0.009, p=0.019, p<0.000) and reduced the live cell population by 23,51%, 22,21%, 54,9%, 41% and 49,81% (p=0.006, p=0.011, p<0.000) for 10nΜ, 50nΜ, 100nM, 200nM and 400nΜ of D/BZ treatments, respectively. Cell cycle profiling also highlighted a much greater sensitivity of Kasumi-1 cells in D/BZ combinations compared to single treatments, with a significant increase in the G1 population by 11,84% (p<0.000), 12,55% (p<0.000), 6,62% (p=0.021), 8,38 (p=0.006) for all D/BZ combinations (from lower to higher) tested, except 400nM D/BZ. A significant decrease in S phase was observed after all combination treatments tested, compared to single ones, by 36,26%, 39,25%, 26%, 30,99% and 30,8% (lower to higher D concentration), (p<0.000), while finally, G2/M population was increased by 56,35% (p<0.000), 85,34% (p<0.000), 34,29% (p=0.02), 81,92% (p<0.000) and 138,45% (p<0.000), respectively.

Conclusions : Our data indicate that the addition of bortezomib -a proteasome inhibitor which, among others, is capable of inducing oxidative stress- to low-dose decitabine significantly enhances apoptosis and decreases live cell population of Kasumi-1, with the combinations of 100nM and 200nM of D with BZ appearing as the most successful ones. Moreover, cell cycle profiling revealed that D/BZ treatment synergistically leads to G1 and G2 arrest, hence prohibiting cells to either synthesize DNA (S phase) or proceed to mitosis. Although these observations need to be further investigated at a molecular level, they appear very encouraging for better understanding the mechanisms underlying primary resistance to decitabine and offer new directions for a much needed, more successful epigenetic therapy in AML.