Role of unphosphorylated STAT5 in maintenance of Acute Myeloid Leukemia cells
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Abstract
Background
In its phosphorylated state, the Signal Transducer and Activator of Transcription 5 (STAT5) A and B form homo- or heterodimers, which bind to chromatin and activate expression of target genes. In leukemia models harboring FLT3-ITD, BCR-ABL or JAK2 mutations, constitutive phosphorylation of STAT5 activates key proliferation and survival transcriptional programs. Recent studies in mouse hematopoietic progenitor cells suggest a distinct function of unphosphorylated STAT5 (uSTAT5): via restricting the access of ERG to target genes uSTAT5 acts as a repressor of megakaryocytic transcriptional programs [1]. The goal of this study is to examine the biological role of uSTAT5A and B in acute myeloid leukemia (AML) and to explore their role as potential therapeutic targets.
Results
We initially screened a panel of human AML cell lines and patient samples for STAT5A and STAT5B expression and phosphorylation at defined tyrosine residues. Most of the samples displayed strong expression of both STAT5A and STAT5B. Phosphorylation of STAT5 proteins at tyrosine 694 (pSTAT5A) and 699 (pSTAT5B) residues was strongly dependent on the presence of FLT3-ITD mutations. To explore the role of uSTAT5A/B, we performed doxycycline-inducible, short-hairpin RNA (shRNA) mediated knock-down of STAT5A and STAT5B. Targeting STAT5A or STAT5B severely suppressed cell proliferation across the entire tested panel; nevertheless, differentiation assays revealed that only the suppression of uSTAT5B induced cellular differentiation. In line, gene expression profiling by high-throughput sequencing (RNA-seq), demonstrated enrichment of monocytic differentiation programs in the THP-1 cell line upon loss of uSTAT5B. To further assess the distinct effects of STAT5A and STAT5B, we performed SILAC-based mass spectrometry and identified several STAT5 interacting partners in AML cell lines. While uSTAT5A primarily was found to be associated with proteins involved in RNA processing and translation initiation pathways, uSTAT5B coprecipitated chromatin- and histone-binding proteins, such as the transcription factor ETV6 or the histone H3K4 demethylase KDM5C.
Finally, to elucidate the role of STAT5 on leukemic cell function in a mouse model, Stat5fl/fl or Stat5fl/fl_Mx1-Cre bone marrow cells were transformed with a retroviral construct of Mll/Af9 and transplanted into lethally irradiated mice. In the second round of transplantations, we performed a pIpC induction of Mx-1-Cre recombinase to explore, whether complete excision of Stat5 can prevent leukemia development. Surprisingly, animals transplanted with Stat5-depleted MA9 cells died briefly after excision with median survival of 15 days, while median survival of the Stat5fl/fl control was 26 days. Bone marrow cells from both cohorts were investigated for the expression of CD11b and Gr-1, both markers of differentiated hematopoietic cells. Interestingly, the Stat5fl/fl_Mx1-Cre-MA9-group, depleted of Stat5, showed a strong increase in the double-positive cells compared to control group suggesting a more mature phenotype.
Summary
In summary, our data indicate that uSTAT5B is involved in the regulation of differentiation through modulation of the epigenetic landscape and transcriptional programs of leukemic cells. Targeting of uSTAT5B or its downstream pathways might represent an interesting novel strategy in AML treatment.