The Jak2 V617F Oncogene Associated with Polycythemia Vera Requires a Functional FERM Domain for Transformation and for Expression of the Myc and Pim Proto-Oncogenes.

The V617F activating point mutation in Jak2 has been shown to be associated with a proportion of patients with myeloproliferative disorders, including polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor (such as the Epo receptor), but the role of receptors in transformation by Jak2V617F has been controversial. Constructs expressing wild type or Jak2V617F, with and without a point mutation in the FERM domain (Y114A) required for receptor binding, were introduced into BaF3 cells already expressing a human Epo receptor. These cells were then evaluated for growth factor independence, response to growth factors, and activation of several critical Jak2 signaling pathways. We found that, whereas BaF3-EpoR cells are readily transformed by Jak2V617F to Epo-independence, the addition of the FERM domain mutation blocked transformation to factor independence. Also, the FERM domain was required for induction of reactive oxygen species by Jak2V617F. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2 V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, Jak2V617F induced expression of the Myc and Pim proto-oncogenes, which are known to cooperate in the transformation of hematopoietic cells. The expression of both proteins was dependent on a functional FERM domain. Finally, we evaluated the role of active STAT5 in transformation of BaF3 cells by introducing a STAT5 mutant that is constitutively activated, and found that activation of STAT5, by itself, was sufficient to induce Jak2V617F-dependent downstream targets believed to be critical for transformation, including Myc and Pim. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5. These results also suggest that small molecules that target Jak2, the FERM domain, or STAT5 would have activity in diseases associated with V617F mutations.