Abstract B009: The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells

Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), currently in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We have reported its strong preclinical activity in lymphomas as a single agent and in combination with several targeted anti-lymphoma agents (AACR 2018, AACR 2019). Here we report the anti-proliferative activities of pracinostat and its primary metabolite SB991 as single agents and in combination with the demethylating agent 5-azacitidine in acute myeloid leukemia (AML) cell lines. Methods. To assess anti-proliferative activity cells were exposed to increasing doses of pracinostat, SB991 (Helsinn) and vorinostat (LC Laboratories) alone or in combination (pracinostat and SB991) with increasing doses of 5-azacitidine (Selleckchem) for 72h, followed by the MTT assay. The Chou-Talalay index was used to determine synergism. Combination indexes (CI) defined the following: additive effect (CI 0.9-1.1), synergism (CI 0.3-0.9) and antagonism (CI > 1.1).For apoptosis and cell cycle analyses cells were treated with 250 nM pracinostat for 72 hours. Results. HL-60 and MV-4-11 cells responded well to pracinostat with IC50 values of 362 nM and 93 nM, respectively. IC50 values obtained for SB991, the main metabolite of pracinostat, were 233 nM and 85 nM for HL60 and MV-4-11, respectively. These IC50 values compared well with those obtained for vorinostat, which equaled 710 nM for HL-60 and 205 nM for MV-4-11. Combination of pracinostat with 5-azacitidine showed synergism in both AML cell lines (CI = 0.51 for HL-60 and CI = 0.69 for MV-4-11). Combination of SB991 with 5-azacitidine achieved synergism in MV-4-11 cells (CI = 0.54) but was not beneficial in HL-60 cells (CI = 1.23). Pracinostat moderately induced apoptosis in HL-60 cells while MV-4-11 cells underwent massive apoptosis. Cell cycle analysis revealed marked G2/M arrest plus moderate accumulation of cells in the sub-G1 phase for HL-60 cells, which was in agreement with the moderate induction of apoptosis measured by Annexin V staining. Also in agreement with the apoptosis analysis, MV-4-11 cells treated with pracinostat all accumulated in the sub-G1 phase. Conclusions. Pracinostat and its main metabolite SB991 exhibited robust anti-proliferative activity as single agents in HL-60 and MV-4-11, comparing favourably with the FDA approved HDACi vorinostat. Pracinostat mediated its anti-proliferative action by inducing apoptosis and G2/M cell cycle arrest. Combination of pracinostat with 5-azacitidine enhanced its activity in AML cells. These results provide pre-clinical rationale for the ongoing phase 3 trials of pracinostat in combination with azacitidine in AML. Citation Format: Afua Adjeiwaa Mensah, Filippo Spriano, Eugenio Gaudio, Annalisa Bonifacio, Emanuela Lovati, Claudio Pietra, Francesco Bertoni. The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B009. doi:10.1158/1535-7163.TARG-19-B009