Detection of Activated STAT5 in the Cytoplasm of Neoplastic Cells in Patients with AML, CML, and Systemic Mastocytosis.
Authors: Sonneck Karoline , Fritz Richard , Muellauer Leonhard , Gleixner Karoline V. , Mayerhofer Matthias , Florian Stefan , Kerenyi Marc , Sperr Wolfgang R. , Sillaber Christian , Moriggl Richard , Valent Peter 
Published: October 2019

The signal transducer and activator of transcription 5 (STAT5) has recently been implicated as essential pro-oncogenic factor in the pathogenesis of myeloid leukemias in mice (Cancer Cell2005;7:87–99). More recently, STAT5 activation has also been described to occur in human leukemias. However, so far, little is known about the expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in various myeloid neoplasms and about the distribution of pSTAT5 in the cellular compartments of the normal and leukemic bone marrow (bm). We have examined the expression of pSTAT5 in the bm in patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6, M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5, blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded bm sections by immunohistochemistry using the pSTAT5-specific antibody AX1. In the normal bm, the antibody AX1 was found to react with megakaryocytes and immature myeloid progenitor cells, whereas erythroid cells and mature granulocytic cells did not stain positive for AX1. In patients with AML and CML, the distribution of pSTAT5 showed a similar pattern. In fact, pSTAT5 was found to be expressed in leukemic blast cells without differences among FAB types as well as megakaryocytic cells, but not in erythroid cells. In patients with SM, neoplastic mast cells were found to be immunoreactive for pSTAT5. Interestingly, in all patients and all cells examined, pSTAT5 was found to be localized in the cytoplasm rather than in the nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was confirmed by immunocytochemical staining experiments performed on primary isolated neoplastic cells (AML, CML, mastocytosis) and respective cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity of neoplastic cells with the AX1 antibody was abrogated by preincubation of the antibody with a pSTAT5-specific blocking peptide. Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell lines was demonstrable by flow cytometry. To study the molecular mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells with doxycycline-inducible expression of disease-specific oncoproteins, namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction of these oncoproteins in Ba/F3 cells resulted in massive activation of pSTAT5 and DNA binding activity as shown by EMSA and supershift assays. In summary, our data show that neoplastic cells in AML, CML, and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins contribute to STAT5-activation. The particular cytoplasmic localization of pSTAT5 in neoplastic cells suggests that apart from its function as a transcription factor, pSTAT5 may have an additional role as a cytoplasmic regulator in these malignancies.