MP-A08 and venetoclax treatment exhibits antileukemic activity in primary AML samples. (A) Primary AML cells were treated with increasing concentrations of MP-A08 for 6 hours, lysed, and subjected to immunoblot analysis with indicated antibodies. (B-C) Primary AML samples were treated with MP-A08 and venetoclax for 6 hours and assessed for cell viability by Annexin V staining. Data are displayed as the mean ± range of duplicate technical replicates. Statistical significance was assessed by Student t test. *P < .05; ***P < .0005. Synergy was determined by the CI method. Fluorescence-activated cell sorting–purified iLSCs were seeded alone (D) or on an MSC coculture layer (E), treated with MP-A08 and venetoclax for 24 hours, and assessed for cell viability by Annexin V staining. Data are displayed as the mean ± range of duplicate technical replicates. Statistical significance was assessed by Student t test. *P < .05. Synergy was determined by the Webb fractional product method. (F) Normal bone marrow–derived CD34⁺ cells were treated with MP-A08 and venetoclax for 24 hours and assessed for cell viability by Annexin V staining. Data are displayed as the mean ± range of duplicate technical replicates. (G) Representative immunohistochemistry staining using human specific mitochondrial antibody (MTC02) of an NSG mouse sternum engrafted with primary AML cells. Bar represents 100 μm. (H-I) NSG mice were engrafted with primary AML blasts and bled weekly to confirm disease engraftment (>1% hCD45⁺ in peripheral blood). Mice received vehicle, MP-A08 (100 mg/kg intraperitoneally), venetoclax (75 mg/kg orally), or both daily for 2 weeks. Engraftment was quantified by assessing the percentage of human CD45⁺ cells in the bone marrow of recipient mice. Each symbol represents the percentage of CD45⁺ cells observed in a separate mouse. Significance was assessed by Student t test. (J) Mutational analysis of AML patient samples treated with venetoclax from the Beat AML Project.⁴⁶ The average area under the curve (AUC) is a measure of drug sensitivity (the higher the AUC, the more resistant) derived from ex vivo drug sensitivity assays. Statistical significance was assessed by Student t test with Welch’s correction. **P < .01; ***P < .0001. (K) Primary AML samples identified by whole-exome sequencing containing PTPN11, TP53 (L), or K-Ras (M) mutations were treated with MP-A08 and venetoclax for 6 hours and assessed for cell viability by Annexin V staining. Data are displayed as the mean ± range of duplicate technical replicates. Statistical significance was assessed by Student t test. **P < .01.