The JAK2V617F-Bearing Vascular Niche Regulates Hematopoietic Stem/Progenitor Cell Regeneration Following Radiation Injury

Abstract

IntroductionMyeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion. Endothelial cells (ECs) are an essential component of the hematopoietic niche. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Previously, we reported that the JAK2V617F-bearing vascular niche promotes the expansion of the JAK2V617F HSPCs in preference to JAK2WT HSPCs. In this work, we test the hypothesis that the JAK2V617F-bearing vascular niche protects MPN HSPCs from radiation injury.MethodsJAK2V617F Flip-Flop (FF1) mice and Tie2-Cre mice were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and ECs (Tie2/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsTo investigate the effects of the JAK2V617F-bearing vascular niche on MPN hematopoiesis in vivo, WT CD45.1 marrow cells were transplanted into lethally irradiated (950cGy) Tie2/FF1 mice or age-matched littermate control mice (CD45.2) (n=12 in each group). During a 3-month follow up, while all WT control recipients displayed full donor engraftment, 7 of 12 Tie2/FF1 recipient mice displayed recovery of the JAK2V617F-mutant hematopoiesis (mixed donor/recipient chimerism) 10 weeks after transplantation. (Figure 1) In contrast to the Tie2/FF1 recipients with full WT donor engraftment, the mixed chimeric mice developed neutrophilia, thrombocytosis, splenomegaly, and significantly expanded CD45.2 JAK2V617F-mutant HSPCs during an 8 month follow up, resembling what we observed in the primary Tie2/FF1 mice in a previous study. These findings suggest that the JAK2V617F-mutant HSPCs in the Tie2/FF1 mice are relatively protected from lethal irradiation.To investigate whether this radioprotection phenotype is due to the JAK2V617F mutation in Tie2/FF1 HSPCs, we generated a chimeric murine model with JAK2V617F-mutant HSPCs and a WT vascular niche by transplanting JAK2V617F marrow cells into WT recipients. The transplantation of WT marrow cells into WT recipients served as a control. Following hematopoietic recovery, each set of mice were irradiated with 300cGy to create a radiation injury, and 2 hr later marrow Lin- HSPCs were isolated for evaluation of cellular apoptosis and cell cycle status. We found that cell apoptosis was significantly increased in the JAK2V617F-mutant HSPCs compared to JAK2WT HSPCs (14.4% vs 2.2%, P=0.035). There was no significant difference in cell cycle status between the JAK2V617F HSPCs and JAK2WT HSPCs. (Figure 2) These data suggest that, in the WT vascular niche, the JAK2V617F-mutant HSPC is more (not less) sensitive to radiation-induced apoptosis than JAK2WT HSPCs.We then investigated whether the hematopoietic radioprotection phenotype is a result of the JAK2V617F-bearing vascular niche protecting HSPCs. Marrow Lineageneg (Lin-) HSPCs were isolated from Tie2/FF1 mice and age-matched littermate controls and cultured on a feeder layer of JAK2WT or JAK2V617F ECs. After 300cGy irradiation, we observed higher numbers of cells from JAK2V617FHSPCs cultured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs (1.6-fold, P= 0.026). In addition, JAK2WT ECs displayed increased cell apoptosis post irradiation compared to JAK2V617F ECs (19.5% vs 7.7%, P=0.026). qRT-PCR of irradiated murine ECs confirmed that both epidermal growth factor (EGF) (4.0-fold, P=0.011) and pleiotrophin (PTN) (11.4-fold, P=0.00001), two niche factors involved in the regulation of HSPC regeneration following radiation injury, were up-regulated in JAK2V617F-bearing ECs compared to JAK2WT ECs. (Figure 3) These results suggest that the JAK2V617F-bearing vascular niche contributes directly to HSPC radioprotection, possibly by its elaboration of soluble niche factors, such as EGF and/or PTN.ConclusionsIn summary, we report that the JAK2V617F-bearing vascular niche protects the JAK2V617F HSPCs from the otherwise lethal irradiation administered during conditioning for marrow transplantation. Together with our previous studies, our findings indicate that the mutant vascular niche could contribute to disease relapse and poor donor cell engraftment following stem cell transplantation in patients with MPNs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.