Methotrexate Is a Suppressor of JAK/STAT Pathway Activation Which Inhibits JAK2V617F Induced Signalling
Authors: Thomas Sally J , Fisher Katherine , Brown Stephen , Snowden John A , Danson Sarah , Zeidler Martin 
Published: October 2019

Abstract

The classical myeloproliferative neoplasms (MPNs) are a group of disorders characterised by activation of the JAK/STAT signalling pathway. A large proportion of patients with MPNs have an acquired mutation, JAK2V617F, which causes constitutive kinase activity. Patients with wild-type JAK2 show gene expression patterns characteristic of JAK/STAT activation, and the majority have mutations in other genes associated with increased pathway activation. Inhibition of JAK/STAT activation represents an attractive therapeutic approach for these disorders. In myelofibrosis, treatment with a JAK inhibitor reduces spleen volume, improves quality of life and prolongs life expectancy, whereas there is evidence that other therapies are no better than placebo.
This study aimed to find new treatments for MPNs by identifying compounds that suppress JAK/STAT activation. We screened a small-molecule library consisting of FDA approved drugs, cytotoxic drugs, agrochemicals, and pure natural products to identify modulators of STAT-responsive transcription in the low complexity Drosophila model. We independently identified methotrexate (z-score -8.72) and the chemically similar aminopterin (z-score -8.2) as strong inhibitors of Drosophila JAK/STAT activation. The suppression of transcriptional reporter activity was dose dependent and was observed following activation of the pathway with the Drosophila JAK/STAT ligand Upd and by the gain of function Drosophila JAK HopTuml. To examine whether these results translated to the more complex JAK/STAT signalling pathway of humans we examined the effect of methotrexate in the Hodgkin Lymphoma cell line HDLM-2, which shows constitutive phosphorylation of several JAK/STAT family members. Immunoblotting for phosphorylated pathway components showed that methotrexate produced a dose dependent reduction in levels of tyrosine phosphorylated STAT5 (Y694), without affecting total STAT5 levels. Methotrexate did not affect phosphorylated proteins in other signalling pathways, including pAKT, p c-Jun or pMAPK.
To examine the potential of methotrexate as a treatment for MPNs, we used the HEL cell line, which is homozygous for JAK2V617F and shows STAT5 phosphorylation that is dependent on JAK2 activity. Immunoblotting showed that methotrexate produced a dose dependent reduction in levels of pSTAT5. Significant suppression of STAT5 phosphorylation was seen following treatment with methotrexate at concentrations equivalent to those measured in the serum of patients taking low dose oral methotrexate (0.4 – 0.8 mM, p<0.001, one-way ANOVA with Dunnett’s multiple comparisons test). Suppression of STAT5 phosphorylation persisted in the presence of folinic acid at concentrations used to prevent methotrexate toxicity, suggesting that the effect on JAK/STAT signalling is not mediated by impairment of folate metabolism. Methotrexate did not completely prevent signalling via the JAK/STAT pathway, as methotrexate-treated cells were still able to phosphorylate STAT5 following stimulation with the EpoR/JAK2/STAT5 pathway ligand erythropoietin. The reduction in STAT5 phosphorylation produced by treatment with methotrexate was comparable to that produced by the JAK1/JAK2 inhibitor ruxolitinib, although ruxolitinib was a more potent inhibitor of pathway activation.
Given that methotrexate is used to treat inflammatory disorders and activation of JAK/STAT signalling is involved in the inflammatory response we examined the effect of methotrexate on rheumatoid arthritis fibroblast-like synoviocytes. Methotrexate also reduced constitutive STAT phosphorylation in these primary cells.
Our results suggest that low-dose oral methotrexate should be investigated as a potential treatment for patients with MPNs. We suggest that methotrexate may suppress pathological over-activation of the JAK/STAT pathway sufficiently to control disease and bring about the benefits demonstrated with specific JAK inhibitors, without preventing physiological activation required for haematopoiesis and response to infection. Methotrexate is inexpensive and has advantages as a potential treatment as its safety and adverse effects are already well understood. Furthermore, our results suggest that suppression of JAK/STAT pathway activation may be a mechanism through which methotrexate exerts anti-inflammatory and immunosuppressive effects.

Disclosures
Off Label Use: Methotrexate is widely used as a chemotherapy drug and to treat inflammatory disorders. This work includes a discussion of the potential use of methotrexate to treat myeloproliferative neoplasms, based on experiments in cell lines.