Combination of 5-Azacytidine and ABT-199 Has a Synergistic Apoptotic Effect in High-Risk MDS/sAML after HMA Failure

Introduction:

Myelodysplastic syndromes (MDS) are one of the most common haematological disorders of the older patient. Allogeneic stem cell stransplantation (ASCT) is the sole curative treatment. The number of eligible patients is limited due to age and comorbidities and only approximately one-third of these patients are cured by ASCT. Further therapeutic strategies such as hypomethylating agents (HMA) have often a short response duration.

We recently found that ABT-199 effectively induces apoptosis in the leukemic progenitor compartment of higher-risk MDS and sAML patients, whereas healthy controls and low-risk patients remain unaffected (Jilg et al., 2016). These data suggest that pro-apoptotic ABT-199 might harbor potential as a novel treatment modality in higher-risk MDS patients. However, it remains unclear whether ABT-199 is a powerful option in patients after HMA failure. Synergistic effects of ABT-199 and 5-azacytidine (5-AZA) were also not analyzed in primary material of MDS/sAML patients yet.

Here we investigate the effect of ABT-199 as single treatment and in combination with 5-azacytdine in bone marrow samples of 28 high-risk MDS/sAML patients under HMA therapy. We measured induction of apoptosis after ABT-199/5-azacytidine treatment and analyzed the effect on colony forming capacity.

Methods:

Purified bone marrow mononuclear cells (BMMNC) were treated with 1μM ABT-199 or a soluble control (DMSO) and/or 5-AZA (from 0.25µM to 10µM) for 72h in vitro. Apoptosis was analyzed by flow cytometry after staining for 7-AAD, Annexin V, and CD34 as a progenitor marker. The long-term survival was investigated by colony formation assay. Control samples were obtained from human femoral heads discarded after implantation of total endoprosthesis of the hip joint from hematologically healthy age-matched donors. Combination indices were calculated using ComboSyn.

Results:

28 primary samples of patients under HMA treatment (complete response n=5; stable disease n=10 and primary failure n=13) were treated with either ABT-199 single and/or combination therapy with ABT-199 and 5-AZA. Combination therapy showed a clear synergistic effect with the most favorable combination index (CI=0.1446) at ABT-199 (1µM) and 5-AZA (1µM). As expected ABT-199 only showed reduced activity in patients with complete remission. However BCL-2 inhibition effectively decreased the number of viable CD34⁺ cells in patients with stable disease under HMA treatment. Patients with HMA have a very poor prognosis. As expected 5-AZA (1µM) had only slight effects on MDS stem/progenitor cells of these patients in the ex vivo setting. ABT-199 monotherapy was still able to effectively induce apoptosis in a 72h read-out. Combination therapy (ABT-199 and 5-AZA at a concentration of 1µM each) efficiently induced apoptosis in the CD34⁺stem/progenitor cells as well as in the bulk of BMMNCs. When compared with single treatment, we found that induction of apoptosis was significantly increased in the stem/progenitor population from patients with HMA failure after combination treatment (ABT-199 mono vs combination p= 0.0089 and 5-AZA mono vs. combination p=0.0006).

Conclusion:

To identify novel treatment options in higher-risk MDS/sAML patients, we analyzed the apoptotic effect of ABT-199 single and ABT-199/5-azacytidine in combination in primary samples of patients under 5-azacytidine treatment.

ABT-199 and 5-azacytidine showed strong synergistic effects with the most favorable combination index with 5-azacytidine (1µM) and ABT-199 (1µM).

Monotherapy with ABT-199 was able to induce cell death ex vivo in high-risk MDS/sAML evenafter HMA failure. Combination therapy induced apoptosis very effectively and significantly reduced colony forming capacity. Age-matched healthy controls were only marginally effected.

We therefore conclude that combination of low-dose 5-azacytidine with ABT-199 is more effective than single treatment in this pre-treated cohort of high-risk MDS/sAML patients. Since patients with HMA failure have a very poor prognosis with limited treatment options combination therapy of ABT-199 and 5-azacytidine seems to be a promising therapy option.