Figure 4.

BCL6 inhibition ablates LSCs. (A) Measurement of expression levels of BCL6 in LSCs (CD45^dim, CD34⁺, and CD38^–) and blast cells (CD45^dim, CD34⁺, and CD38⁺) were measured by intracellular staining and flow cytometry. Averages of absolute mean fluorescence intensity (MFI) values of PE-BCL6 are shown from triplicates, with standard deviations as error bars. (B) Representative flow cytometry data of the analysis done in (A). (C) Three different primary AML cells were treated with 10 μM RI-BPI for 48 hours, and percentages of dead cells were measured by using annexin V–fluorescein isothiocyanate (FITC) staining and flow cytometry. P values indicate significance of correlations between dose and percent live cells for each of the primary AML cells. (D) Representative flow cytometry density plots to compare untreated and RI-BPI treatment. (E) Primary AML cells were treated with 10 μM RI-BPI for 24 hours in duplicates, and colony numbers were counted after 14 days. Values were normalized to vehicle-treated controls. The data are plotted as mean of duplicates. Error bars indicate standard deviation. (F) Schematic illustration and (G) comparison of results in 2 different in vivo behaviors of AML cells treated with a BCL6 inhibitor. Two primary AML cells (AML2 and AML9) were subjected to 24 hours of ex vivo pretreatment with either RI-BPI (5 μM), control peptide inhibitor (CPI), or were untreated (UT). The treated cells were then injected into patient-derived xenograft recipient mice. Six to 8 weeks after the injection, percentages of human AML cells were monitored by measuring mCD45^–, hCD45⁺, and hCD33⁺ cells in the BM aspirates. Schematic illustration (H) and comparison of results (I) of in vivo engraftment of AML cells transfected with BCL6 siRNA. BCL6 or nonspecific control siRNA transfected AML cells (AML33) were injected in mice. The plot shows the percentages of human AML cells in the presence or absence of BCL6 knockdown. Rx, radiation.