JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 ^V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 ^V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 ^V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 ^V617F clones.

We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 ^V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 ^V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 ^V617F cells to outcompete WT cells in mouse transplant experiments.

From these mouse data, we hypothesized that Jak2 ^V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 ^V617F. We found that Jak2 ^V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 ^V617F clones (but never IL-10Rα Jak2 ^WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 ^V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 ^V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 ^V617F has transforming ability. Together, these data support our hypothesis that Jak2 ^V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway.

It has been previously shown that JAK1 can transactivate JAK2 when JAK2 ^V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 ^V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 ^V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 ^V617F and JAK1 are currently ongoing.