The Role of STAT5 in HOXA9-Associated Leukemia

Abstract

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that is essential for hematopoietic stem cell expansion and differentiation. Deregulation of HOXA9 is commonly observed in human acute myeloid leukemia (AML). About half of AML patients overexpress HOXA9 as a result of MLL rearrangements, NUP98 translocations, NPM1 mutations or CDX2/CDX4 overexpression. Despite its central importance in leukemia, the mechanism of transcriptional regulation by HOXA9 and its downstream effectors are poorly understood. HOXA9 physically interacts with MEIS1, a cofactor that greatly accelerates leukemia development in transplanted animals. Our group recently identified a number of transcription factors as HOXA9 potential collaborators by genomic profiling of HOXA9 binding sites and mass spectroscopy. One of these putative collaborators is signal transducer and activator of transcription 5 (STAT5), which coimmunoprecipitates with HOXA9. Furthermore STAT motifs extensively overlap with HOXA9 binding sites. STAT5 is important for survival, proliferation and differentiation of hematopoietic cells and constitutive activation of STAT5 has also been observed in human leukemias bearing oncogenic mutation of Jak2, Bcr-Abl, c-Kit and Flt3. FLT3 internal tandem duplication (FLT3-ITD) is observed in 25% of patients with MLL-partial tandem duplication (MLL-PTD) and is associated with HOXA9 upregulation and unfavorable prognosis. Therefore, we hypothesized that the interaction of HOXA9 and STAT5 may play a role in HOXA9-associated leukemogenesis.Treatment of human cell lines bearing MLL-AF9 and FLT3-ITD with specific FLT3 and STAT5 inhibitors showed that suppression of the constitutive activation of STAT5 significantly inhibits the hyper-proliferation of these cells. We then overexpressed FLT3-ITD or active mutation of STAT5 (STAT5 1*6) in mouse hematopoietic stem cells /progenitor cells (HSC/PCs) transduced with MLL-AF9 or HOXA9 and found that constitutively active STAT5 enhances cell proliferation in vitro. We next transduced HOXA9 into HSC/Pcs from wild type (WT) or FLT3-ITD transgenic mice and transplanted these cells into sublethally irradiated WT mice. All of these recipients developed myeloid leukemia, with recipients transplanted with FLT3-ITD (n=4) developing leukemia significantly earlier than WT controls (n=5, p<0.05), suggesting that FLT3-ITD mediated STAT5 activation enhanced HOXA9-induced leukemogenesis in vivo.To further assess the role of STAT5 in HOXA9-mediated transformation, we performed ChIP-Seq assay with HOXA9-transformed cells and identified nearly half of STAT5 binding sites (228 out of 596) colocalized with HOXA9. Most of these cobound sites are located in distal intergenic (61.0%) and intron (35.1%) regions. Five cobound regions (Il2rα, Fgf1, Pdlim5, Pim1, Fabp5) were selected and confirmed by ChIP-qPCR. To further characterize the interaction between HOXA9 and STAT5, GST pull-down assays were performed that showed that the c-terminal of HOXA9 is critical for interaction with STAT5. Overall, the findings suggest that STAT5 promotes HOXA9-induced transformation by functionally interacting with HOXA9 at HOXA9-regulated enhancers. DisclosuresNo relevant conflicts of interest to declare.