Uncovering Non-Canonical Signalling Mediated By JAK2V617F in Myeloproliferative Neoplasms
Authors: Wadley Lucas , Lai Hew Yeng , Arora Eshika , Huang Helen , Heidmann Jianhong C , Jing Dennis , Chen Lauren , Fleischman Angela G. 
Published: December 2024

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in hematopoietic stem cells (HSCs) that leads to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2V617F, leading to constitutive JAK-STAT signalling and excessive proliferation of myeloid cells. In humans, for MPN to arise, JAK2V617F HSCs must have a selective advantage over WT hematopoietic stem cells (HSCs). However, in mouse MPN models, Jak2V617F 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 Jak2V617F clones. Chronic inflammation plays a central role in the development and progression of myeloid malignancies. Many studies have shown chronic exposure to an inflammatory agent such as lipopolysaccharide (LPS) in murine models results in chronic HSC proliferation with subsequent exhaustion. Given this inflammatory environment, it is essential to understand the mechanisms that regulates inflammatory responses in MPN. One key player in this regulatory process is interleukin-10, an anti-inflammatory cytokine that helps maintain inflammatory homeostasis.

We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic rather than acquired abnormality. In mice, we found that blockade of IL-10R prolongs cycling of WT HSCs but not Jak2V617F HSCs after an acute exposure to LPS. Additionally, using H2B GFP mice we measured the proliferative history of HSC chronically exposed to LPS +/- IL-10R blocking antibody for two weeks. HSC from mice treated with the combination of LPS + IL-10R blocking antibody underwent more divisions compared to mice treated with LPS alone. Utilizing image-flowcytometry (IFC), HSCs exposed to chronic LPS + anti-IL-10R exhibit decreased levels of polarity, a feature of HSC exhaustion. Consistent with this, bone marrow from mice exposed to chronic LPS + IL-10R performed inferiorly to bone marrow from mice exposed to LPS alone in competitive repopulation assays.

With defective IL-10R signalling, WT HSCs are ill equipped to thrive in the inflammatory niche of MPN. However, we found that blockade of IL-10R allows Jak2V617F cells to outcompete WT cells in mouse competitive transplants. These in vivo results suggest that the defective IL-10R signalling may contribute to the expansion of JAK2V617F cells and acts as a driver of MPN disease progression. Although JAK2 canonically does not participate in the IL-10R pathway, we hypothesized that JAK2V617F enhances IL-10R signalling. To test this, we created Ba/F3 cell lines co-expressing IL-10R and JAK2V617F. We found that JAK2V617F enhances growth at limiting concentrations of IL-10 and results in cytokine independence with long latency in culture, consistent with the hypothesis that JAK2V617F enhances IL-10R signalling. Western blot analysis showed that in Ba/F3 cells co-expressing IL-10R and JAK2V617F, not only was the IL-10R pathway activated through pSTAT3 in the absence of cytokine, endogenous JAK1 was also activated. Through a receptor pulldown assay, we detected JAK2 associated with IL-10R complex binding in JAK2V617F expressing cells only. We hypothesize that JAK2V617F is utilising the mechanism of transactivation to force the IL-10R signal to occur through mediation of JAK1. Preliminary data from a receptor pulldown assay of mIL-10R did show JAK2V617F presence, along with JAK1 and STAT3 when stimulated with IL-10, but not endogenous wild-type JAK2. Like IL-10R signalling, IFNa signalling doesn't use JAK2, so a JAK1 deficient U4C cells were used to study JAK1 mediated signalling via IFNa stimulation to determine JAK1's necessity for cytokine independent JAK2V617F signalling activation. Preliminary data is showing JAK2V617F expressing U4C cells activating STAT1 even in the absence of JAK1, a part of the IFNa pathway, co-IP and receptor pulldown assays are currently ongoing to determine JAK2V617F and IFNa receptor association.