Fig. 8: Fine-tuned expression of LRC regulators is required for leukemia progression.

a Experimental design for competitive transplantation of JUN-overexpressing-patient leukemia cells. Doxycycline-inducible JUN or TagBFP-transduced, mCherry-expressing patient leukemia cells are separately engineered and isolated by fluorescence-activated cell sorting. Equal cell numbers of JUN and TagBFP-transduced cells are transplanted into NSG mice with or without doxycycline diet in vivo. b Leukemia-free survival of mice transplanted with a mixture of JUN and TagBFP-transduced MSK165 patient leukemia cells with or without doxycycline diet is shown (25,000 cells/mouse, 11 mice for each group). (Supplementary Fig. 12a, b for MSK011 and MSK162). c–e Genomic DNA of engrafted patient leukemia cells is analyzed to determine the dominant clones of propagated leukemia cells in each mouse (refer to Supplementary Fig. 12c–f). Stacked bar charts represent the proportion of dominant clones in each group with (bottom bar) or without (upper bar) doxycycline induction in MSK165 (left), MSK011 (center) and MSK162 (right). JUN-transduced clones are relatively reduced upon doxycycline induction, exhibiting enforced JUN expression impairs leukemia progression (28.6% to 0%%, 33.3% to 0%, and 62.5% to 33.3% for MSK165, MSK011, and MSK162 leukemias, respectively). f Experimental design to investigate leukemia progressing property of JUN-knockout patient leukemia cells, which are generated using Cas9 crRNA;ATTO550-labeled-tracrRNA RNP electroporation, isolated by fluorescence-activated cell sorting, and transplanted into NSG mice. g Leukemia-free survival of mice transplanted with JUN-knockout or control AAVS1-targeted MSK165 patient leukemia cells (30,000 cells/mouse, 15 mice for each group), where JUN-knockout cells propagate leukemia with similar kinetics as control AAVS1-targeted cells (log-rank p = 0.74 and 0.85 for JUN gRNA-1 and gRNA-7 versus AAVS1, respectively). (Supplementary Fig. 14b–e for MSK011 and MSK162). h Mutant allele frequencies are analyzed using the Tracing of Indels by Decomposition (TIDE) method before and after transplantation of JUN-knockout or control AAVS1-targeted MSK165 patient leukemia cells. JUN-knockout clones are enriched upon leukemia progression in vivo, whereas AAVS1-targeted cells are not (n = 10, 9 and 8 mice; two-tailed Welch’s t test p = 0.031, 0.064 and 0.21 for JUN gRNA-1, gRNA-7 and AAVS1, respectively). a, f Free illustration materials from Kenq Net (https://www.wdb.com/kenq/illust/mouse) and SciDraw (https://doi.org/10.5281/zenodo.4152947 and https://doi.org/10.5281/zenodo.5204473) are used. Source data are provided as a Source Data file.