Figure 2.

Combination of SY-1425 and hypomethylating agents result in apoptosis and DNA damage in RARA-high and IRF8-high cells. Induction of apoptosis was measured via increase in caspase 3/7 with Promega™ Caspase-Glo® 3/7 Assay Systems in RARA-high AML cell lines (A) OCI-AML3 and (B) MV;4-11 as well as (C) RARA-low AML cell OCI-M1 and (D) Kasumi-1 pretreated with either DMSO, azacitidine, decitabine, or RG108 for 24 hours, followed by 24 Hours with SY-1425. There was a statistically significant increase in apoptosis induction (P<0.001 as measured by a two-tailed t-test) in cells treated with combination versus either SY-1425 alone or HMA alone, where the degree of response was related to the RARA expression. Upon addition of SY-1425 to cells treated with HMAs, DNA damage was detected via western blot based on induction of pH2A.X and PARP cleavage in (E) RARA-high OCI-AML3, and (F) RARA-low OCI-M1 AML cell lines. OCI-AML3 showed synergistic increase in pH2A.x only in the combination of azacitidine or decitabine with SY-1425 but not with RG108. Contrastingly, OCI-M1 show high pH2A.X with treatment of azacitidine or decitabine alone, which was only slightly increased when combined with SY-1425. Data shown is representative of results of three separate experiments. AZA: Azacitidine; DEC: Decitabine; SA: Single Agent.