Secondary shRNA screen for genes required by MLL-AF9 AML in vivo. (A) Schematic of secondary screen. MLL-AF9 transduced mouse granulocyte monocyte progenitors were serially transplanted into recipient mice. Quaternary transplanted leukemia cells (c-Kit^hiCD34⁺FCRγRII^hi) were sorted and infected with 3 pools of shRNAs in 5 replicates and transplanted into sublethally irradiated mice. Pretransplant cells (day 0 sample) and day 14 bone marrow samples were sequenced to determine shRNA representation. (B) Positive control shRNAs are significantly more depleted than negative control shRNAs. Shown is the median log₂ fold depletion of 5 replicates. Lines indicate mean. ***P < .001, *P < .05, t test. (C) List of 13 screen hits.