BACKGROUND: Venetoclax, a small molecule BH3 mimetic which inhibits the 
anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have 
both shown activity as single-agent treatments in pre-clinical and clinical 
studies in acute myeloid leukemia (AML). In this study, we deliver the rationale 
and molecular basis for the combination of idasanutlin and venetoclax for 
treatment of p53 wild-type AML.
METHODS: The effect of idasanutlin and venetoclax combination on cell viability, 
apoptosis, and cell cycle progression was investigated in vitro using 
established AML cell lines. In vivo efficacy was demonstrated in subcutaneous 
and orthotopic xenograft models generated in female nude or non-obese 
diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action 
analyses were performed by means of cell cycle kinetic studies, RNA sequencing 
as well as western blotting experiments.
RESULTS: Combination treatment with venetoclax and idasanutlin results in 
synergistic anti-tumor activity compared with the respective single-agent 
treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly 
superior efficacy in vivo, in subcutaneous and orthotopic AML models. The 
inhibitory effects of idasanutlin were cell-cycle dependent, with cells 
arresting in G1 in consecutive cycles and the induction of apoptosis only 
evident after cells had gone through at least two cell cycles. Combination 
treatment with venetoclax removed this dependency, resulting in an acceleration 
of cell death kinetics. As expected, gene expression studies using RNA 
sequencing showed significant alterations to pathways associated with p53 
signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin 
treatment. Only few gene expression changes were observed for venetoclax 
treatment and combination treatment, indicating that their effects are mediated 
mainly at the post-transcriptional level. Protein expression studies 
demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to 
the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in 
response to combination treatment contributing to the superior combined 
activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown 
studies.
CONCLUSIONS: Our findings provide functional and molecular insight on the 
superior anti-tumor activity of combined idasanutlin and venetoclax treatment in 
AML and support its further exploration in clinical studies.