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K-Ras mutation depletes pre-leukemic HSC
between samples was determined using an analysis of variance (ANOVA) for multiple comparisons following Tukey’s multiple comparisons test.
Results
Aml1-ETO ameliorates key features of the myeloproliferative neoplasm phenotype caused by K-RasG12D
To study the effect of combining Aml1-ETO with mutant K-Ras, whilst avoiding the previously described spontaneous recombination in primary KrasG12D/+;Mx1- Cretg/+ mice,19,20 we generated E14.5 FL cells with the neces- sary genotypes: Aml1ETO/+;Mx1-Cretg/+ (AM genotype); KrasG12D/+;Mx1-Cretg/+ (KM genotype); Aml1ETO/+;KrasG12D/+;Mx1-Cretg/+ (AKM genotype); and Mx1- Cretg/+ controls (CON genotype); all CD45.2 allotype. FL cells were used as these have been previously shown to have either no or minor spontaneous recombination using Mx1-Cre.29 To create a scenario where competitive advan- tage and disadvantage of mutant HSC could be observed, mice were generated where the hematopoietic system was partially repopulated by experimental cells and par-
A
tially by WT competitor cells (CD45.1 allotype). This was done by competitively transplanting 2.5x105 FL cells (CD45.2) and 1x106 WT BM competitor cells (CD45.1) into lethally irradiated CD45.1 recipients, as previously described.27 Recombination was induced four weeks post- transplantation by subcutaneous injection of poly(I:C), with all groups of mice treated, including controls. Long- term monitoring of primary transplanted mice was not possible as both KM and AKM developed a T-cell leukemia (data not shown), as previously reported for KM.20 Therefore, the hematopoietic phenotype was analyzed eight weeks post-poly(I:C) before T-cell leukemia devel- opment occurred (Figure 1A).
AM-transplanted recipients displayed mild leukopenia compared to CON-transplanted mice (Figure 1B), due to a decrease in B and T cells in the peripheral blood (PB) (Online Supplementary Figure S1A and B), whereas no differ- ence in hemoglobin levels, BM cellularity, Mac1+Gr1lo myeloid cells in the PB or spleen, or spleen weight was observed (Figure 1C-H). However, AM showed a 27% decrease in platelet count compared to CON (Figure 1I). These results suggest Aml1-ETO affects B and T cell, as well as platelet development, in line with known func- tions of AML1 (also known as RUNX1).30,31
Figure 1. Aml1-ETO ameliorates the key features of the myeloproliferative neoplasm phenotype caused by K-RasG12D. (A) Schematic representation of in vivo competitive transplant experiment. (B-I) Analysis of recipients of Mx1-Cretg/+ controls; CON (n=13), Aml1ETO/+;Mx1-Cretg/+; AM (n=14), KrasG12D/+;Mx1-Cretg/+; KM (n=12) and Aml1ETO/+;KrasG12D/+;Mx1-Cretg/+; AKM fetal liver (FL) (n=14) for (B) peripheral blood (PB) white blood cell (WBC) count. (C) PB hemoglobin levels. (D) Bone marrow (BM) cellularity per tibia and femur. (E and F) CD45.2 Mac1+Gr1lo myeloid cells as absolute number in the PB (E) and spleen (F). (G) Representative FACS plots of Mac1+Gr1+
and Mac1+Gr1lo myeloid cells as LiveCD19–CD4–CD8a–NK1.1–CD45.2+ cells across all experiments in the PB. (H) Spleen weight. (I) Platelet count. Results were generated in three independent experiments. The results were analyzed using multiple com- parison ANOVA and are presented as mean±Standard Deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
a percentage of
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