PU-H71 effectively induces cell death in TP53-mutant AML cells and stem/progenitor cells and prevents the outgrowth of TP53-mutant AML cells. (A) TP53-WT and TP53-R248W Molm13 cells were treated with PU-H71 or BH3 mimics targeting either BCL-2 (venetoclax [VEN]) or MCL-1 (AZD5991). (B-D) TP53-WT, -KO, and -mutant Molm13 cells (B), TP53-mutant primary AML cells and stem/progenitor cells (C), and healthy BM and BM stem/progenitor cells (D) were treated with PU-H71. After 48 hours of treatment, cell death and cell counts were determined via flow cytometry. Primary cells were cocultured with MSCs. (E-F) A mixture of GFP-labeled TP53-WT and BFP-labeled TP53-R248W Molm13 cells (1000:1 ratio) was treated with nutlin3a or PU-H71 (E) and flow cytometry was used to assess cell death and surviving TP53-WT (GFP) and TP53-R248W (BFP) cells or treated with VEN or PU-H71 (F), and cell viability and cell numbers were assessed via flow cytometry in the presence of counting beads. Fresh drugs and cell culture medium were added 2 or 3 times per week. 7AAD, 7-aminoactinomycin D; AnnV, annexin V; VEN, venetoclax.