Mutations that disrupt DNA or CBFβ binding impair AML1-ETO's leukemogenic activity. (A) Schematic of transplantation scheme. BM mononuclear cells harvested from 5-FU–treated C57BL/6 mice were co-infected with MigR1 expressing AML1-ETO (and its mutated derivatives) and TEL-PDGFβR. Internal ribosome entry site (IRES)–mediated expression of EGFP marks AML1-ETO expressing cells, while hCD4 marks TEL-PDGFβR expressing cells. Retrovirally transduced cells (10⁶) were transplanted along with 2 × 10⁵ normal BM cells into lethally irradiated mice. (B) FACS plots of BM cells 2 days after transduction to show that all were equivalently infected. (C) Kaplan-Meier survival curve of mice after transplantation with retroviruses expressing AML1-ETO (AE) or its mutated derivatives and TEL-PDGFβR (TP). The study end point was 63 days. The number of mice in each group is indicated. (D) Spleen weights of mice upon sacrifice. Significant differences from AML1-ETO plus TEL-PDGFβR recipients (#) are indicated with * (Dunnett test and ANOVA). (E) Representative plots of BM cells in transplant recipients upon sacrifice demonstrating the presence of EGFP⁺ and hCD4⁺ cells. The relatively high EGFP fluorescence in the AML1-ETO (Y113A/T161A) plus TEL-PDGFβR BM cells was observed in 19 of 24 mice. (F) Average percentage of EGFP⁻ hCD4⁺ and EGFP⁺ hCD4⁺ BM cells in transplant recipients upon sacrifice. Error bars indicate 95% confidence intervals. Differences relative to AML1-ETO plus TEL-PDGFβR (#) for each group were determined using Dunnett test and ANOVA. (G) Cytospin preparations of BM cells from an AML1-ETO plus TEL-PDGFβR leukemic mouse purified based on hCD4 and EGFP expression.