Epigenetic silencing mediated by non-phosphorylated STAT5B prevents differentiation in acute myeloid leukemia

STAT5 proteins are transcription factors involved in the regulation of proliferation, apoptosis and differentiation. Upon ligand-mediated activation, phosphorylated STAT5A (pSTAT5A) and pSTAT5B form homo- or heterodimers, translocate to the nucleus and activate transcription. Recent data indicate an important role of un-phosphorylated STAT5 (uSTAT5) in the regulation of differentiation. For example, the presence of uStat5 shifts the balance toward the maintenance of leukemic stem cells, whereas genetic depletion of Stat5 induced differentiation. Further, uSTAT5 has been shown to act as a repressor of megakaryocytic differentiation via restricting the access of ERG to its target genes. To further investigate the function of uSTAT5 in mammalian AML, we established several cell line models expressing doxycycline-inducible shRNA directed against either STAT5A or STAT5B. In uSTAT5-expressing AML cells targeting of STAT5A or STAT5B suppressed cell growth and induced differentiation. These effects were significantly stronger upon knockdown of STAT5B compared to STAT5A. Global RNA sequencing demonstrated enrichment of differentiation programs upon downregulation of uSTAT5B. To further explore the biological function of uSTAT5, we performed SILAC-based global proteomics after pulldown of either STAT5A or STAT5B. While uSTAT5A was found to be associated with proteins involved in RNA processing, uSTAT5B primarily co-precipitated with chromatin- and histone-binding proteins, like the transcriptional repressor ETV6 or the histone H3K4 demethylase KDM5C. To dissect the specific roles of pSTAT5B and uSTAT5B, we performed co-immunoprecipitation assay upon treatment of AML cells with GM-CSF or vehicle control. As expected, GM-CSF treatment caused strong phosphorylation of STAT5A/B, however, uSTAT5B-binding of ETV6 was almost completely abolished, indicating a specific association only with uSTAT5B. As depletion of uSTAT5B induced differentiation and uSTAT5B interacts with KDM5C and ETV6, we hypothesized that uSTAT5B prevents transcription of genes involved in differentiation. To address this question, we performed H3K4me3 ChIP-seq analysis in THP-1 cells upon knockdown of STAT5B. Downregulation of uSTAT5B caused strong accumulation of H3K4me3 at promoter regions of previously silenced genes. Finally, we wanted to investigate whether targeting of KDM5C can reverse disturbed differentiation in AML cells. Treatment with the KDM5 inhibitor CPI-455 significantly inhibited proliferation and induced apoptosis, but only in AML samples expressing uSTAT5B. In summary, our data provide evidence that uSTAT5B specifically blocks differentiation in AML cells via its interaction with KDM5C followed by repression of H3K4me3 at distinct promoter regions. Targeting of uSTAT5B or its interacting partners might represent an interesting novel strategy in AML therapy. Citation Format: Jakub Szybinski, Daniel Sasca, Jan Heidelberger, Karolin Klumb, Viral Shah, Anna Dolnik, Matthias Theobald, Lars Bullinger, Petra Beli, Thomas Kindler. Epigenetic silencing mediated by non-phosphorylated STAT5B prevents differentiation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5223.