Conditional repression of NRAS^G12V expression after the establishment of AML results in remission, prolong survival, and relapse. (A) Serial transplantation of previously engrafted AML BM cells into secondary recipient SCID mice. The secondary recipient SCID mice were treated with Dox either chronically or for 6 hours at a time after the establishment of full-blown AML. (B) Kaplan-Meier survival curve of all AML2 and AML4 transplantations shows that both chronic and temporary Dox treatments significantly increase the survival time compared with those transplanted mice not treated with Dox after the establishment of AML (P < .001). The arrow indicates the time that chronic or 6-hour Dox treatment started. (C) WBC concentration in the peripheral blood of the secondary recipient SCID mice that were transplanted with AML2 BM cells from TRM-transgenic mice. The first Dox in the arrowed square means the start of chronic and temporary (6-hour) Dox treatments. Other Dox in the arrowed squares indicated the repeated 6-hour Dox treatment. Chronic Dox in the top panel means the continuous Dox treatment instead of the 6-hour Dox treatment was started at this time point. In the top panel, n = 4 were treated with chronic and n = 3 with 6-hour Dox. In the bottom panel, n = 2 were treated with chronic, n = 2 with 6-hour, n = 4 with no, and n = 3 (Mll-AF9 only) with chronic Dox. Narrow bars show standard deviations for WBC concentration at each measurement. (D) Flow cytometry of spleen cells from the engraftment, remission, and relapse in secondary recipient SCID mice. The spleen of engrafted mice contains greater than 50% of myeloid (Mac1/Gr1⁺) lineage. After 8 days of chronic Dox treatment, most GFP-positive cells are eliminated. However, after relapse, the spleen contains more than 70% GFP/Mac1 or GFP/Gr1 double-positive cells. (E) Western blot analysis for transgenic NRAS^G12V and endogenous Nras proteins. Due to the EE tag at the N terminus of NRAS^G12V, the molecular weight of NRAS^G12V is approximately 1 kDa larger than endogenous Nras. Both NRAS^G12V and endogenous Nras proteins are decreased at day 4 and not detected at day 8 after chronic Dox treatment. GAPDH is used as a loading control.