In the previous funding period, we have generated a novel model of JAK2V617F-positive erythropoiesis using immortalized I/11 mouse erythroid progenitor cells which have been shown to faithfully execute essential steps of erythropoiesis. Three major results have been achieved during the previous funding period: (1) of particular interest was the finding that expression of JAK2V617F mutated kinase in hematopoietic cell lines is sufficient to directly induce expression of a number of pro-inflammatory cytokines including IP-10, TNF-α, and IL-6; (2) PLCγ1 was identified as a master signaling node in function and differentiation of EpoR/JAK2 controlled erythropoiesis; (3) in preliminary experiments, we found that expression of JAK2V617F induces dramatic dysregulation of integrin (LFA1, VLA4) expression, adhesion and polarization on ICAM-1 and VCAM-1. Moreover, additional results indicate a strong synergism of LPS-induced Toll-like receptor (TLR) signaling with JAK2V617F in induction of the pro-inflammatory chemokine/cytokine IP-10. This may contribute to the cytokine storm observed in patients. Interestingly, this hypothesis is supported by the finding that IP-10 is significantly up-regulated in primary myelofibrosis patients and independently predictive of inferior survival.
In the next funding period, I/11 cells expressing either JAK2WT or JAK2V617F will be employed as our main in vitro model. In addition, we will take advantage of a conditional JAK2V617F knock-in mouse model made available through our collaborators. Employing these tools, we aim to characterize the role of JAK2V617F in regulating integrin (LFA1, VLA4) activation and function in vitro and in vivo. Specific findings will be validated in primary human cells (granulocytes, monocytes, B-cells, T-cells) from CMN patients. We also seek to identify the signaling molecules connecting JAK2V617F with integrin signaling. To gain a comprehensive view on the role of PLCγ1 in JAK2V617F-induced inflammation in vivo we will generate a conditional PLCγ1 knock-out/JAK2V617F knock-in mouse. Using this model, we will study inflammatory cytokines in granulocytes, T-, B-cells and serum, integrin adhesion and function and the inflammation-related disease phenotype (splenomegaly, myelofibrosis, extramedullary hematopoiesis). Finally, we will dissect at a molecular level the cooperation of TLR signaling with JAK2V617F signaling for induction of the pro-inflammatory chemokine/cytokine IP-10.