INKA1-OE induces label retention and prolonged LSC latency in vivo. Transduced CD34⁺CD38⁻ 8227 AML cells were expanded for 2 (EXP1), 6 (EXP3), or 9 weeks (EXP2); labeled with CellTrace Far Red; and transplanted into NSG mice (10⁶ cells/mouse, EXP1: n = 2; EXP2: n = 4-5; EXP3: n = 13-15 for 3 analysis time points: 4, 8, and 12 weeks). (A) Human CD45⁺ cell engraftment at 4 weeks. (B) BFP⁺(CD45⁺) engraftment relative to %BFP⁺(CD34⁺) input at 4 weeks shown as mean per experiment. (C-F) Freshly isolated BM from individual mice was pooled per group and experiment, mouse cell depleted and analyzed for CD34/CD38 expression and label retention (CellTrace). (D) The relative abundance of CD34⁺CD38⁻ cells in CTRL/INKA1 transduced cells (BFP⁺) vs BFP⁻ cells within the same pools was assessed. (E-F) CellTrace label retention in BFP⁺ cells. Student t test. (G) CD45⁺ engraftment for EXP3: 3 time points for primary transplants (4, 8, 12 weeks) and 2 secondary transplantation experiments from 8 and 12 weeks CD45⁺BFP⁺ pooled and sorted primary grafts (additional 8 weeks: 8 weeks + 8 weeks, 20 000 cells/mouse; 12w+8w, 100 000 cells/mouse) were analyzed. (H) Longitudinal analysis of percent CD34⁺ in BFP⁺ fraction. Secondary transplants are connected to the point of their primary transplantation origin by a dotted line. Student t test with Welch’s correction. (I) Cell cycle analysis from mouse cell depleted and CD45⁺BFP⁺ sorted cells from 8 and 12 weeks primary transplantation. (J) BFP⁺ percentage within CD45⁺ population for each individual mouse and time is shown next to input (%BFP⁺[CD34⁺]). (K) H4K16ac staining from mouse cell depleted and CD45⁺BFP⁺ sorted cells from 8 and 12 weeks primary transplantation. (L) Shown is the CD34⁺/CD15⁺ composition of the CD45⁺BFP⁺ graft of individual mice after secondary transplantation (12 weeks + 8 weeks). *P < .05.