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Figure 2. The JAK2V617F-mutant hematopoietic stem/progenitor cell (HSPC) is more sensitive to radiation-induced apoptosis than JAK2WT HSPC. (A) Outline of experiment design to generate a chimeric murine model with JAK2V617F-mutant HSPCs and wild-type (WT) vascular niche. (B) After 300cGy irradiation, cell apoptosis (Annexin V+7AAD–) was significantly increased in JAK2V617F Lin– HSPCs (black bar) compared to JAK2WT Lin– HSPCs (gray bar). (C) Unirradiated JAK2V617F (black) Lin– HSPCs proliferated to a greater extent than JAK2WT (gray) Lin– HSPCs in vitro. (D) Peripheral blood donor chimerism following a competitive repopulation assay in which 5x105 post-irradiated JAK2WT or JAK2V617F marrow cells (CD45.2) were injected together with 1x105 competitor CD45.1 WT marrow cells into lethally irra- diated CD45.1 WT recipients (n=4 in each group). *P<0.05.
JAK2V617F-bearing vascular niche in MPNs
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lethal irradiation administered during conditioning for marrow transplantation. To confirm this hypothesis, we irradiated primary Tie2/FF1 mice or Tie2-cre control mice with 300cGy, and two hours later their marrow Lin- HSPCs were isolated for evaluation of cellular apoptosis (Figure 1F). We found that the JAK2V617F HSPCs in the mutant vascular niche (i.e. Tie2/FF1 mice) had significant- ly less cellular apoptosis compared to JAK2V617F HSPCs in WT vascular niche (i.e. control mice) (12.1% vs. 25.8%; P=0.043) (Figure 1G). Therefore, the JAK2V617F-mutant HSPCs in Tie2/FF1 mice are relatively protected from lethal irradiation, which could be responsible for the reported high incidence of disease relapse in patients undergoing allogeneic SCT for MPNs.2,6,7
The JAK2V617F-mutant HSPC is more sensitive to radiation-induced apoptosis than JAK2WT HSPC
Tie2-Cre mice express Cre recombinase in both ECs and hematopoietic cells. To investigate whether the radiopro- tection phenotype noted in the prior experiments is due to the JAK2V617F mutation in Tie2/FF1 HSPCs, we generat- ed a chimeric murine model with JAK2V617F-mutant HSPCs and a WT vascular niche by transplanting Tie2/FF1 marrow cells into WT recipients. The transplantation of WT marrow cells into WT recipients served as a control. Following hematopoietic recovery and full donor cell engraftment, each set of mice were again irradiated with 300cGy to create a radiation injury, and two hours later, marrow Lineageneg (Lin-) HSPCs were isolated for evalu- ation of cellular apoptosis and cell cycle status. In our pre- vious study, recipient mice of Tie2/FF1 marrow developed a MPN phenotype by eight weeks post transplantation with significant thrombocytosis and neutrophilia.19 Therefore, we irradiated the mice at six weeks post trans- plant in this study before the development of any clinical phenotype (data not shown) (Figure 2A). We found that cell
apoptosis was significantly increased in the JAK2V617F- mutant HSPCs compared to JAK2WT HSPCs (47.3% vs. 20.3%; P=0.005) (Figure 2B). This result is consistent with the observation that unirradiated JAK2V617F Lin– HSPCs proliferate to a greater extent than JAK2WT Lin– HSPCs in serum-free medium in vitro (5.8-fold; P=0.000006) (Figure 2C), and therefore are predictably more sensitive to radia- tion-induced apoptosis. There was no significant differ- ence in cell cycle status between the JAK2V617F HSPCs and JAK2WT HSPCs after irradiation. These data suggest that, in the WT vascular niche, the JAK2V617F-mutant HSPC is more (not less) sensitive to radiation-induced apoptosis than are JAK2WT HSPCs.
To further test the effect of irradiation on HSPC func- tion in the WT vascular niche, we performed a competi- tive repopulation assay in which 5x105 post-irradiated marrow cells (CD45.2 JAK2WT or CD45.2 JAK2V617F) were injected intravenously together with 1x105 competi- tor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients (Figure 2A). Since the presence of JAK2V617F mutation in HSPCs may affect the cell’s long- term proliferation, we focused on donor cell chimerism in the early phase of engraftment. During an 8-week post- transplant follow up, there was no difference in CD45.2 donor chimerism between the recipients of post-irradiated JAK2V617F marrow cells and recipients of post-irradiated JAK2WT marrow cells, suggesting that the engraftment potential of post-irradiated JAK2V617F HSPCs (or at least the short-term HSPCs) do not differ from JAK2WT HSPCs (Figure 2D).
JAK2V617F-bearing ECs protect HSPCs from lethal irradiation
We next studied the effects of an EC JAK2V617F muta- tion on hematopoietic radioprotection. Lin- marrow HSPCs were isolated from WT or Tie2/FF1 mice and cul-
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