Extended Data Fig. 10: Treatment with a PARP1 inhibitor suppresses in vivo induction of leukaemia in mice co-transplanted with NK cells, but does not affect expression of NKG2DLs in healthy haematopoietic cells and baseline haematopoiesis.
From: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion
a, b, Treatment with AG-14361 in vitro (pre-transplantation) or in vivo (post-transplantation) inhibits the capacity for the in vivo initiation of leukaemia of human AML cells in NSG mice co-transplanted with NK cells. a, In vitro pre-treatment: AML cells were cultured in vitro with AG-14361 (20 μM) or DMSO (0.2%) for 24 h and then transplanted via tail vein injection into NSG mice (1.5 × 106 AML cells per mouse), which afterwards were co-transplanted (or not) with pNKCs (1.5 × 107 pNKCs per mouse) (n = 3 mice per condition and patient, n = 5 cases of AML). Mice were analysed for the presence of leukaemic cells in bone marrow, peripheral blood, liver and spleen at 16 h after transplantation. Summarized results of n = 5 independent biological experiments are shown, after normalization to ‘DMSO control without NK cells’ (which was set to 1). Statistical analysis was performed using a two-sided Mann–Whitney U test. b, In vivo treatment: mice transplanted with human AML cells (no. 35, 1.5 × 106 AML cells per mouse, transplanted intrafemorally, n = 6 mice per group) were treated with AG-14361 (10 mg kg-1 intraperitoneally, on days 1 to 5 after transplantation) or DMSO control, and afterwards co-transplanted (or not) with pNKCs (4.5 × 106 pNKCs per mouse administered once intravenously on day 6 after transplantation, pre-treated or not with anti-NKG2D or isotype control, 5 μg ml-1). Mice were analysed at week nine after transplantation for leukaemic engraftment. Note that between day 6 after transplantation and the final analysis time point, no further treatment was applied. See schematic of the experiment and further data with higher (10:1) pNKC:AML cell ratio in Fig. 4j, k. Statistical analyses were performed using a two-sided Student’s t-test. c–i, Surface expression of NKG2DLs on healthy human (c–f) and mouse (g–i) haematopoietic cells at baseline and after PARP1 inhibition. Representative flow cytometry data of the indicated human haematopoietic cells derived from peripheral blood (c) and bone marrow and cord blood (d–f) of healthy donors, and quantification (f). In g, expression of NKG2DLs is quantified on different subpopulations of healthy mouse haematopoietic bone marrow cells (n = 3 mice; bulk leukaemic cells from Mll-Enl and Mll-ptd/Flt3-ITD mice are shown as positive controls). h–j, Treatment of mice with AG-14361 (10 mg kg-1, intraperitoneally) or DMSO vehicle control, and subsequent quantification of expression of NKG2DLs on haematopoietic cells. Schematic of the experiment (h) and quantification of percentages of NKG2DL+ cells among total mouse haematopoietic cells of specific compartments (i) (haematopoietic stem cells (HSCs): KIT+Lin−SCA1+CD150+CD48; MEP, megakaryocyte–erythroid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte–macrophage progenitor) and absolute numbers of white blood cell counts (j) (with distribution of neutrophils, lymphocytes and monocytes) and red blood cell counts (n = 3 mice per group). Centre values represent mean, error bars represent s.d. Two-sided statistical tests were performed using Mann–Whitney U test (a) or Student’s t-test (b, f–j). Centre values represent mean, error bars represent s.d. for all plots.
