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. 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 × 10 6 AML cells per mouse), which afterwards were co-transplanted (or not) with pNKCs (1.5 × 10 7 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 × 10 6 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 × 10 6 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.
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