HIF1α and colony-forming properties of the relapsed AML cells. (A) Relapsed MllPTD/WTFlt3ITD/WT AML cells contain of a small subset of cells with constitutive HIF activity under normoxia. Relapsed AML cells were transduced with a lentiviral reporter in which the expression of GFP is under the control of hypoxia-responsible element (HRE, 5′-GTA CGT GAC CAC-3′); a lentiviral vector in which the GFP expression is controlled by a mutated HRE (HRE-MT, 5′-GTA AAA GAC CAC-3′) was used as control. The leukemia cells were analyzed by fluorescence-activated cell sorter at 72 hours after transduction. (B) Echinomycin effectively eliminates the subset of relapsed AML cells with HIF activity under normoxia. As in A except the relapsed AML cells were cultured in the presence of given concentration of echinomycin as indicated. (C) Transduction efficacy of scrambled shRNA (Sh-Srambled) or Hif1α shRNA (Sh-Hif1α) for the relapsed AML cells. Lentiviral vectors expressing either Sh-Scrambled or Sh-Hif1α in conjunction with GFP were transduced into the leukemia cells, and the transduction efficiencies were assayed at 72 hours after transduction. (D) Sh-Scrambled or Sh-Hif1α transduced cells from relapsed MllPTD/WTFlt3ITD/WT AML described in C were assayed for LIC activities by CFU assay. Hif1α shRNA silencing of HIF1α reduces LIC activity within relapsed MllPTD/WTFlt3ITD/WT AML, as measured by CFU activity and compared with control. (E) Sh-Scrambled or Sh-Hif1α transduced blasts from relapsed MllPTD/WTFlt3ITD/WT AML described in C were assayed for LIC activities by CFU assay. In this dose escalation experiment, silencing Hif1α reduces the sensitivity of relapsed MllPTD/WTFlt3ITD/WT AML LIC to echinomycin as the dose of echinomycin is increased. The relapsed AML cells from C were treated for 24 hours with the noted concentrations of echinomycin before plating the cells into metrogels for CFU assay. Data shown in D and E are means ± standard deviation (SD) of triplicate cultures. All data in this figure have been reproduced twice.
