Figure 1

Sustained suppression of NR4A1/3 is required for AML maintenance and NR4A rescue is sufficient to deplete AML LICs. (a) Kaplan–Meier plot shows Cre⁺NR4A3^−/−NR4A1^fl/fl (conditional-DKO) mice die from disease between 10–20 weeks post Tam injection, whereas appropriate control mice remain healthy until cessation of the study. (b) Peripheral blood, bone marrow and spleen cytospins show greater than 20% immature blasts, a defining feature of AML. Original magnification is × 40. Scale bars=50 µM. (c) Retroviral NR4A3 rescue scheme. (d) Kaplan–Meier plot of lethally irradiated CD45.1 mice transplanted with CD45.1 donor cells, plus leukemic whole bone marrow from conditional-DKO mice (CD45.2) treated with pMIG-Empty (green), or pMIG-NR4A3 (pink) retrovirus. Two million transduced GFP⁺ cells were transplanted along with competitor CD45.1 cells at an 8:1 ratio. As controls, lethally irradiated CD45.1 mice were also transplanted with competitor cells or wild-type (WT) cells, and all of these mice showed no sign of AML development and survived until study cessation (not shown). (e, f) Donor CD45.2-derived AML cell engraftment in transplanted mice shows that CD45.2⁺ NR4A3-GFP (green) expressing cells are eliminated over time in contrast to GFP control leukemic cells, which continue to expand until the animals die. The inability of NR4A-rescued LICs to sustain long-term engraftment prevents AML maintenance. Even at an 8:1 CD45.2:CD45.1 ratio at transplantation, wild-type CD45.1 cells out-compete for long-term engraftment, implying that NR4A reactivation leads to loss of LIC self-renewal/stemness.