title: Methotrexate Is a JAK/STAT Pathway Inhibitor.
abstract: BACKGROUND
  The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains.
  
  
  RESULTS
  We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin.
  
  
  CONCLUSIONS
  Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential.
authors.affiliation: The Bateson Centre, and The Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, United Kingdom; Academic Unit of Clinical Oncology, The University of Sheffield, Sheffield, S10 2TN, United Kingdom.
authors.forename: Sally
authors.initials: S
authors.lastname: Thomas
authors.affiliation: The Bateson Centre, and The Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, United Kingdom.
authors.forename: Katherine H
authors.initials: KH
authors.lastname: Fisher
authors.affiliation: Academic Unit of Clinical Oncology, The University of Sheffield, Sheffield, S10 2TN, United Kingdom; Department of Haematology, Royal Hallamshire Hospital, Sheffield, S10 2JF, United Kingdom.
authors.forename: John A
authors.initials: JA
authors.lastname: Snowden
authors.affiliation: Academic Unit of Clinical Oncology, The University of Sheffield, Sheffield, S10 2TN, United Kingdom; Sheffield Experimental Cancer Medicine Centre, Weston Park Hospital, Sheffield, S10 2SJ, United Kingdom.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Danson
authors.affiliation: The Sheffield RNAi Screening Facility, Department of Biomedical Science, The University of Sheffield, Firth Court, Sheffield, S10 2TN, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Brown
authors.affiliation: The Bateson Centre, and The Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, United Kingdom.
authors.forename: Martin P
authors.initials: MP
authors.lastname: Zeidler
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology*; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D004791:Enzyme Inhibitors; D050799:STAT5 Transcription Factor; D053614:Janus Kinase 2; D008727:Methotrexate