Activation of AP-1 By JAK2V617F through Induction and Stabilization of c-FOS
Authors: Edahiro Yoko , Araki Marito , Hironaka Yumi , Yang Yinjie , Morishita Soji , Sunami Yoshitaka , Ohsaka Akimichi , Komatsu Norio 
Published: October 2019

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, has been detected in most patients with Philadelphia chromosome (Ph)-negative classical myeloproliferative neoplasms (MPNs). The identification of the JAK2 mutation in MPNs led to the development of JAK2 inhibitors, which suppress the symptoms of this disease. However, treatment with these inhibitors rarely leads to molecular remission and is associated with side effects caused by blockade of function of wild-type JAK2. In addition, resistance has been reported; therefore, a new therapeutic strategy based on understanding of MPN development is required. Here, we found that JAK2V617F dramatically upregulates cytomegalovirus (CMV) promoter activity in HEK293T cells. By performing a reporter assay using luciferase, we found that a non-canonical AP-1 binding motif is responsible for this activation of the CMV promoter. By performing a series of biochemical analyses, including an electrophoresis mobility shift assay and a supershift assay with antibodies, we found that c-FOS, a member of the AP-1 family of proteins, bound to oligo duplex DNA containing the identified motif only when JAK2V617F was expressed. A reporter construct harboring the identified sequence alone showed a robust increase in reporter assay when JAK2V617F was induced, while mutation of the AP-1 binding motif abolished the response to JAK2V617F. The results showed that c-FOS overexpression alone is sufficient for activation of the promoter containing the non-canonical AP-1 binding site, demonstrating that c-FOS is a downstream target of JAK2V617F in CMV promoter activation. The activation of this reporter construct was also observed in leukemia cell lines UT-7/EPO and UT-7/GM when JAK2V617F is expressed, which implies that this activation is not limited to HEK293T cells. We found that the activation of AP-1 by JAK2V617F was partly due to robust induction of c-FOS mRNA (41 fold) followed by increase in c-FOS protein levels. In addition, we found that, the ubiquitin-proteasome protein degradation pathway responsible for c-FOS degradation was perturbed in HEK293T cells expressing JAK2V617F, which at least partly contributed to c-FOS protein accumulation and subsequent AP-1 activation. Multiple inhibitors of known downstream pathways of JAK2, such as the mitogen activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways, were analyzed using the reporter assay. The results showed that none of them suppressed JAK2V617F-dependent reporter activation. Furthermore, inhibition of the STAT pathway by expression of the dominant-negative form of STAT1, STAT3, or STAT5 failed to suppress AP-1 activation by JAK2V617F. Consistent with this observation, expression of the active form of STAT1, STAT3, and STAT5 did not affect AP-1 activity. Taken together, we conclude that AP-1 activation by JAK2V617F through c-FOS induction involves an unidentified molecular mechanism. Although our observation is limited to an in vitro system, our finding suggests that deregulation of AP-1 plays a role in MPN development. The AP-1 reporter assay system that we have established should be useful not only for screening novel compounds for MPN treatment but also in studying the novel pathway regulated by JAK2V617F, which would lead to the development of a novel therapeutic strategy against MPNs.

Disclosures

No relevant conflicts of interest to declare.