rhIGFBP7 activates ATRA-driven responses in primary non-APL AML cells. For all ex vivo experiments, cells were stimulated with PBS (Control/Ctrl), 100 µg/mL rhIGFBP7, 0.5 µM ATRA, or the combination (Combi) for 7 days. Percentages of CD33⁺CD11b⁺, viable CD45^dim and CD45^dimCD34⁺ cells were measured using flow cytometry, quantified relative to flow count beads, normalized against untreated control cells, and plotted as mean ± SEM. Patient sample characteristics are summarized in supplemental Table 1. (A) CD11b membrane expression on CD33⁺ blasts relative to the untreated control sample in AML13. (B) Percentage of viable CD45^dim cells (blue; upper panels) and CD45^dimCD34⁺ cells (red; lower panels) in AML7. (C) Heat map of clinical and genetic features of 28 primary AML samples. Responsive AML cases were defined as >5% increase in CD11b-expressing myeloid CD33⁺ blasts and/or >5% reduction in CD45^dim blast survival upon ATRA-rhIGFBP7 combination treatment compared with the single treatments. The fold increase in CD11b and fold decrease in CD45^dim cells represent the ratio of CD11b-expressing CD33⁺ blasts and the ratio of reduction in CD45^dim blast survival following combination treatment relative to untreated control or single therapies. Percentages are shown in supplemental Table 2. (D) Induction of differentiation in 8 primary AML samples responsive to rhIGFBP7-ATRA. Percentage of viable CD45^dim cells (E) and CD45^dimCD34⁺ cells (F) in 10 primary AML samples responding to rhIGFBP7-ATRA. (G) Schematic overview of the experiment (left panel). After injection of T-cell–depleted primary AML cells, NSG mice were treated with ATRA (10 mg, 21-day-release pellet) in week 2 and/or rhIGFBP7 (12 mg/kg) in week 5 (days 1-3). At week 16, the bone marrow cells of the mice were analyzed for the presence of hCD45⁺ cells (middle panel) and myeloid hCD45⁺CD33⁺ cells (right panel) using flow cytometry. *P < .05, **P < .01, ***P < .001, ****P < .0001, 1-way ANOVA with post hoc Tukey’s multiple-comparison test. BM, bone marrow; N/A, not available; PB, peripheral blood.