Figure 6. Targeting GABPβ1L/β2 impairs LSC self-renewal and synergizes with imatinib therapy to eradicate CML.

(A) Intrinsic requirement of GABPβ1L/β2 for LSC survival. Fifteen to twenty days after transplantation of p210^BCR-ABL-infected cells, BM cells from the primary recipients were surface-stained to identify LSCs (GFP⁺ LSKs) and further stained for AnnexinV and 7-AAD. The percentage of AnnexinV⁺7-AAD⁻ apoptotic cells is shown in representative contour plots and summarized in bar graph on the right (n = 4 for control, and n = 8 for dKO). **, p<0.01. (B) LSCs lacking GABPβ1L/β2 exhibit more active cycling. BM cells isolated from the primary recipients as in (A). To avoid quenching of GFP in LSCs, the cells were first incubated with Hoechst33342 at 37^oC for 45 min, followed by two washes with HBSS and surface staining on ice. Percentages of cells in G0/G1 or S/G2/M phases are shown in representative histograms and summarized in bar graph on the right (n = 4 for Ctrl, and n = 8 for dKO). **, p<0.01. (C) Enumeration of LSCs in primary recipients. Primary recipients of p210^BCR-ABL-transduced BM cells were sacrificed on day 15 post-BMT, and GFP⁺ LSK cells in the BM were determined. Representative data from 3 independent experiments are shown. ***, p<0.001. (D) CML propagation in secondary recipients. GFP⁺ LSK cells were sorted from primary recipients of p210^BCR-ABL-transduced BM cells on day 15 post-BMT. Ten thousand sorted cells were injected into another cohort of syngeneic mice along with 2 × 10⁵ protectors, and CML progression was monitored. Data in (D) and (E) are pooled results from 2 independent experiments, and statistical significance was assessed using log-rank test. (E) Synergistic effect of GABPβ1L/β2 deficiency and imatinib therapy in controlling CML in vivo. Primary recipients of p210^BCR-ABL-transduced BM cells were untreated or treated with imatinib (100 mg/kg body weight, b.i.d.) from day 8 to day 80 post-BMT, with CML progression monitored.