Cytoplasmic localization of phosphorylated STAT5 in human acute myeloid leukemia is inversely correlated with Flt3-ITD

To the editor:

The fms-related tyrosine kinase-3 internal tandem duplication (Flt3-ITD) confers growth-factor independence on acute myeloid leukemia (AML) cells and is associated with an unfavorable prognosis. Signal transducer and activator of transcription 5 (STAT5) is a downstream mediator of Flt3 signaling in AML; however, flow cytometry for phosphorylated STAT5 (pSTAT5) did not show specific association with Flt3-ITD.¹  Since a recent study published in Blood by Harir et al²  reported pSTAT5 in the cytoplasm of leukemic samples, we wanted to determine whether this localization to the cytoplasm might be influenced by Flt3-ITD status and whether nuclear pSTAT5 would be a more informative end point. Using a routine immunohistochemistry technique that we previously reported for detecting activated STAT5 in chronic myeloproliferative disease,³  we have now analyzed pSTAT5 staining in myeloid blasts in bone marrow biopsies from patients with AML.

STAT5 tyrosine phosphorylation was detected immunohistochemically with the anti-phosphoSTAT5a/b (Y694/99) mouse monoclonal antibody (AX1; Advantex Bioreagents) as described by others in breast cancer.⁴  In our study, we analyzed a total of 40 patients with AML for nuclear versus cytoplasmic staining of pSTAT5 using the AX1 antibody. Total pSTAT5 was seen in 15 of 40 patients, while the Flt3-ITD was detected in 8 (20%) patients. Interestingly, as shown in Figure 1, Flt3-ITD⁺ samples had predominant nuclear pSTAT5, as would be expected from increased transcriptional activation. However, the Flt3-ITD⁻ samples showed some nuclear staining, but had much more prominent cytoplasmic pSTAT5 staining with significant variation between groups (Kruskal-Wallis test; P = .03). A total of 2 patients showed predominant cytoplasmic staining and no nuclear staining. The distribution of cytoplasmic staining was significantly different between Flt3-ITD⁺ and Flt3-ITD⁻ patients (Mann-Whitney test; P = .04).

The study by Harir et al²  demonstrated in mouse and human samples that STAT5 may potentially have cytoplasmic functions that promote PI3-kinase activation of Akt. Our data confirm the cytoplasmic localization of pSTAT5 in a larger set of patient samples and further suggests that this localization is modulated by the presence of Flt3-ITD. This is an important finding since it suggests that competing biological mechanisms are responsible for the nuclear versus cytoplasmic distribution of pSTAT5 in AML. Cytoplasmic pSTAT5 may not be directed efficiently to the nucleus in the absence of essential interactions provided by the Flt3-ITD scaffold. Alternatively, the hyperphosphorylation of STAT5 mediated by Flt3-ITD may override other mechanisms that may sequester pSTAT5 in the cytoplasm.⁵  This finding could be analogous to the requirement for JAK2^V617F to interact with type I cytokine receptors to promote STAT5 transcriptional activation and cytokine-independent growth.⁶  Finally, our data also demonstrate that detection of nuclear, but not total pSTAT5 could be the most meaningful test for rapidly identifying patients who may respond to Flt3 or other tyrosine kinase inhibitors.