Figure 1.
PAR1 is expressed in hematopoietic cells.
1A. PAR1 was analyzed in mRNA microarray expression data from FACS sorted bone marrow cells [22], [23]. Highest expression was found in hematopoietic stem cells (HSC) and cells of the erythroid/megakaryocyte and of the T-cell lineage. Shown here are log arbitrary units. 1B. Left-hand side: To sort for the different murine bone marrow subpopulation, total bone marrow was stained with lineage-markers, sca1 and c-kit. Lineage-negative, sca1+, c-kit+ (LSK) cells were further divided into long-term (LT)-HSCs as Flt3−CD34− population, short-term (ST)-HSCs as Flt3−CD34+ cells and multipotent progenitors (MPPs) as Flt3+CD34+ cells (upper panel). Common lymphoid progenitors (CLPs) were defined as lineage-negative, IL7R+c-kit+ cells. Upper and lower right panel: Par1 mRNA expression was determined by real-time quantitative RT-PCR using cDNA from the FACS-sorted murine bone marrow subpopulations and Par1 expression was normalized to GAPDH expression. Par1 was expressed in all hematopoietic stem/progenitor subpopulations and CD3+ T-cells whereas monocytes/macrophages/granulocytes (CD11b+) or erythrocytic (Ter119+) or B-cells (B220+) expressed low or no Par1.
Figure 2.
PAR1 function in proliferation and differentiation.
2A. Stem cell FACS analysis revealed similar numbers of stem and progenitor subpopulations (n = 3 mouse pairs for each FACS). Shown here are the percentage of lin−sca1+c-kit+ (LSK) cells from the lineage-negative parental population and the percentage of longterm (LT)-HSCs, shortterm (ST)-HSCs and multipotent progenitors (MPP) from the parental LSK population. 2B. Colony assays of cells from Par1+/+ and Par1−/− total bone marrow (left-hand side) and c-kit+ bone marrow cells (right-hand side; n = 3 mouse pairs for each experiments). No significant changes in the ability of forming colonies were observed in any cell population. 2C. Left-hand side: Bone marrow cells from CD45.2+ Par1−/− or Par1+/+ mice were mixed with Par1+/+ bone marrow from congenic CD45.1+ as depicted in the schematic overview. Right-hand side: At 16 weeks after transplantation, the number of negative responders, which were transplanted with Par1−/− bone marrow, did not differ from wild type transplanted mice. Therefore, the stem cell frequency was comparable in the bone marrow of both genotypes. 2D. Schematic outline of the transplantation experiment using PAR1-overexpressing lineage-negative bone marrow cells compared to control cells transduced with the empty vector. 2E. Colony formation of bone marrow cells transduced with empty vector (“control”) or with a PAR1 expressing retroviral vector (“pMY-PAR1”) was not significantly different between the two groups. 2F. Transplantation of cells as depicted in Figure 2D lead to a significantly lower ratio of PAR1-overexpressing cells after four weeks.
Table 1.
Blood parameters of wild type and Par1−/− mice.
Figure 3.
PAR1 mRNA expression in primary patient samples.
PAR1 expression (3A) was significantly down-regulated in bone marrow cells from human Acute Myeloid Leukemia (AML; n = 67) patients compared to sorted CD34+ cells (n = 5) in microarray analysis, while the expression of PAR2 only showed a non-significant trend (3B), and the expression of PAR3 (3C), PAR4 (3D) and Thrombin (3E) was unchanged. Shown here are log arbitrary units. 3E. PAR1 expression was significantly downregulated in bone marrow cells from human Acute Myeloid Leukemia (AML) patients compared to CD34-positive bone marrow cells. PAR1 expression was determined by qRT-PCR and normalized to GAPDH expression level.
Figure 4.
PAR1 expression in primary patient samples.
4A. Micrographs of Tissue Array analysis from NBM and AML patients stained with anti-PAR1 antibody and Fast-Red secondary antibody contrasted with hematoxylin and eosin. Overview (upper panel) and magnification of one example of CD34+ and AML samples that were defined PAR1-negative (lower left) and PAR1-positive (lower right). 4B. Quantitative Tissue Array analysis of PAR1 expression using categories of staining intensity as positive or negative. Significantly more AML patient samples were negative for PAR1 expression than CD34+ healthy patient samples (p = 0.003, Chi-square test). 4C. PAR1 protein was significantly less abundant in bone marrow cells from human Acute Myeloid Leukemia (AML) patients compared to CD34-positive bone marrow cells in Tissue Array samples. *p<0.05, Chi-square test.
Figure 5.
Absence of Par1 accelerates MLL-AF9 driven murine leukemogenesis.
5A. Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from Par1+/+ or Par1−/− mice was retrovirally transduced with MLL-AF9/GFP. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses and subsequent serial transplantations. 5B. Survival curves of recipient mice which were transplanted with bone marrow cells of Par1+/+ or Par1−/− mice that were retrovirally transduced with MLL-AF9 (n = 8 of each genotype). Cells of both genotypes led to a fatal leukemic disease with comparable latency. 5C. Survival curves of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in Fig. 5B. The secondary recipients of Par1−/−;MLL-AF9 cells (n = 14) died after a significantly shorter latency than mice transplanted with Par1+/+;MLL-AF9 primary blasts (n = 15; p<0.001). 5D. The phenotypic analysis of blasts of the secondary leukemic mice did not reveal differences in CD11b expression between Par1+/+;MLL-AF9 and Par1−/−;MLL-AF9 cells. 5E. Par1−/−;MLL-AF9 transplanted mice (n = 8) exhibited a strong tendency towards higher percentages ofc-kit expressing cells in spleens (p = 0.055, t-test) and bone marrow (p = 0.22, t-test)compared to Par1+/+;MLL-AF9 transplanted mice (n = 4).
Figure 6.
Leukemia initiating cells are regulated by Par1.
6A. For a cloning efficiency assay, Par1+/+;MLL-AF9 or Par1−/−;MLL-AF9 bone marrow cells from leukemia-transplanted mice were FACS-sorted and 1 to 300 c-kit+GFP+ cells were seeded in semi-solid medium in a 48-well plate. Par1+/+ cells had a clone forming frequency of 1/3.4, while the frequency was much higher in Par1−/− cells (1/1.7; p = 0.047). Shown here are the mean results of three independent experiments. 6B. Schematic overview about the serial transplantations performed with MLL-AF9 leukemic blasts. 6C. Left-hand side: Kaplan-Meier plot illustrates the leukemia-free survival of tertiary transplanted mice. After tertiary transplantation, transplantation of 100 MLL-AF9 c-kit+ leukemic blasts revealed a significant elongated life span of mice transplanted with Par1+/+ cells (n = 12) compared to Par1−/− cells (n = 6; p = 0.002). Transplantation of 1000 cells did not reveal significant difference concerning the overall survival. Right-hand side: The frequency of leukemia-initiating cell was calculated according to the results shown in the left-hand plot by using the program L-Calc. Par1+/+ leukemia-initiating cells appeared with a frequency of 1/216, while the frequency was much higher in Par1−/− cells (1/56; p = 0.0166). 6D. Overexpression of PAR1 in Par1−/− MLL-AF9 leukemic spleen cells extents the life time of transplanted mice. Left-hand side: Schematic outline of the experimental of the transplantation. Right-hand side: Kaplan-Meier plot reveals the significant longer latency of leukemia in mice transplanted with Par1-deficient compared to wild type MLL-AF9 c-kit+ blasts overexpressing PAR1.