PIK-75 suppresses Mcl-1 through the inhibition of the transcriptional kinases, Cdk7 and Cdk9, and a block in MCL-1 gene transcription. (A) HEK293T cells were cotransfected with constructs expressing the Renilla luciferase gene (TK promoter-driven) and the Firefly luciferase gene (MCL-1 promoter-driven). Cells were treated with DMSO (vehicle), LY294002 (20 μM), PIK-75 (100 nM), PI-103 (100 nM), IC87114 (5 μM), TGX-221 (1 μM), AS252424 (100 nM), SNS-032 (10 μM), or flavopiridol (10 μM) and the relative luciferase activity was determined after 20 hours. Error bars represent standard errors with asterisks indicating P < .05. (B) The binding affinity of PIK-75 to purified recombinant Cdk7 (data not shown), Cdk9, and Cdk2 was measured in the KINOMEscan competition assay (see supplemental Methods). (C) MV4;11 cells were treated with DMSO, LY294002, flavopiridol, SNS-032, or PIK-75 for 8 hours following which cell lysates were immunoblotted with the indicated antibodies. (D) Bax^−/−Bak^−/− FDM cells were incubated with the indicated drugs following which cell lysates were immunoblotted. (E) Primary AML mononuclear cells were treated with DMSO or PIK-75 (100 nM) for 8 hours following which cell lysates were immunoblotted. Results are typical of at least 2 experiments.