Treatment of SALL4 peptide in primary human AML cells induces reduced cell viability in culture and impaired leukemic engraftment in vivo. (A) wt peptide treatment reduces cell viability of human AML cells in culture and this effect can be reversed in part by the PTEN inhibitor SF1670. A total of 1 × 106 AML cells were left untreated (None); treated with Pep-1 alone (Mock); treated with 20μM wt or scr peptide; treated with wt peptide + 400nM PTEN inhibitor SF1670 (wt + inh); or treated with 100nM TSA for 48 hours, as described in “Methods.” Cell viability of untreated primary AML cells is set as 100 (n = 3 biologic samples). Error bars indicate SD. (B) Schematic diagram showing the steps and time course of the peptide treatment in the xenotransplantation assay. One million primary human leukemic cells were treated twice with peptides at a 24-hour interval, followed by transplantation into sublethally irradiated NSG mice. Mice were killed when they became ill. (C) Wright-Giemsa staining of a cytospin preparation of BM from scr- or carrier alone–treated mice shows marked expansion of immature blasts, which is not present in the wt-treated recipient BM (left panel) Scale bar indicates 10 μm. The scr- or Pep-1 carrier alone–treated mice also have enlarged spleens (middle panel) and kidneys (right panel), whereas the wt-treated recipient mice show normal spleens and kidneys. (D) Histology section of the kidney (arrow) shows effacement of normal architecture by leukemic infiltration. Scale bar indicates 20 μm. (E-F) SALL4 wt peptide treatment significantly impaired human AML cell engraftments in NSG mice analyzed at 78 days after transplantation. The percentage of human CD45+ cells engrafted in the BM, spleen, and peripheral blood was determined by flow cytometry. Representative FACS results from wt, scr, and Pep-1 carrier–only (mock) treatments are shown in panel E and a statistical summary is shown in panel F (n = 5 mice per group). P < .01 in BM; P < .05 in the spleen and peripheral blood by ANOVA with the Tukey multiple comparison test.
