STAT5 Requires the N-Domain for Suppression of miR15/16, Induction of Bcl-2, and Survival Signaling in Myeloproliferative Disease.
Authors: Li Geqiang , Miskimen Kristy L. , Wang Zhengqi , Xie Xiu Yan , Brenzovich Jennifer , Ryan John J , Tse William , Moriggl Richard , Bunting Kevin D. 
Published: October 2019

Abstract
Abstract 211
Phosphorylated signal transducer and activator of transcription 5 (STAT5) can inform diagnosis and clinical outcome in myeloid leukemia. STAT5 may be an important therapeutic target for hematologic disease characterized by constitutively active tyrosine kinases. The most direct evidence that STAT5 is crucial for oncogenic transformation has come from mouse models, which showed that STAT5-deficient hematopoietic cells are resistant to transformation by oncogenic tyrosine kinases, such as TEL-JAK2, TEL-PDGF, or BCR-ABL. The oncogenic role of STAT5 has been recognized and likely extends to other activated kinases such as JAK2, MPL, and FLT3. Although up-regulation of genes such as bcl-2, bcl-XL, mcl-1, D-type cyclins, and myc by activated oncogenic tyrosine kinases has been demonstrated, the specific mechanisms how STAT5 can critically control pre-leukemic expansion in the myeloid lineage have not been defined. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5aS711F) requires the N-domain but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5abnull/null primary mast cells that STAT5a lacking the N-domain (STAT5aΔN) ineffectively protected against cytokine withdrawal-induced cell death relative to wild-type STAT5a. To study the mechanisms for this survival defect, bcl-2 and bcl-XL protein levels were analyzed by Western blot and shown to be greatly reduced. Whether bcl-2 is a direct target gene of STAT5 in native chromatin as validated by chromatin immunoprecipitation (ChIP) has not been previously shown. We identified 7 conserved STAT5 binding sites in the bcl-2 gene and tested these by ChIP. Only one site located within intron 2 was bound by STAT5a and STAT5aΔN in mast cells cultured in IL-3 alone, where bcl-2 mRNA levels were low. STAT5a add-back induced bcl-2 mRNA (10-fold) compared with STAT5aΔN (2- to 3-fold). Interestingly, when STAT5 was absent the mRNA levels of bcl-2 were not reduced when mast cells were grown in the presence of both IL-3 and SCF, despite the virtual absence of bcl-2 protein. MicroRNAs (miRs) are small, noncoding, single-stranded RNAs of ∼22 nucleotides that negatively regulate gene expression at the post-transcriptional level primarily through targeting the 3'-UTR of target mRNAs. There are no current reports of STAT5 mediated miR expression. We found that wild-type STAT5a and STAT5aS711F suppressed accumulation of miR15b/16 in primary mast cells and transduced BaF3 cells. Importantly, we show binding of STAT5 to a conserved STAT5 binding site in the promoter of miR15b/16 by ChIP. We propose that reciprocal induction of bcl-2 mRNA and suppression of miR15b/16 maintained bcl-2 protein levels. We also observed binding of STAT5aΔNS711F at both sites by ChIP, indicating that co-activator and co-repressor interactions with the N-domain determine regulation of bcl-2. To test whether these observations of bcl-2 regulation were physiologically relevant in the setting of activated STAT5 in myeloproliferative disease, retroviral complementation of STAT5abnull/null fetal liver cells was performed and followed by transplantation into lethally-irradiated recipients. Persistently active STAT5aS711F lacking the N-domain (STAT5aΔNS711F) was insufficient to protect c-Kit+Lin−Sca-1+ (KLS) cells from apoptosis as determined by Annexin V/DAPI staining. These cells were unable to induce bcl-2 expression determined by intracellular flow cytometry 15 days following injection of donor cells. In contrast, STAT5aS711F caused robust KLS cell expansion, induction of bcl-2, and 47-fold expansion of peripheral Gr-1+Mac-1+ cells. In the absence of the STAT5 N-domain a mild splenomegaly was observed with 50% of mice surviving greater than 90 days, instead of a rapidly lethal monocytic disease observed when full-length STAT5aS711F was present, with death of all mice by 35 days. Importantly, the modest 6-fold increase in peripheral Gr-1+Mac-1+ counts and better survival conferred by STAT5aΔNS711F could be reversed to 41-fold above control levels by adding back only bcl-2 through H2k/bcl-2 transgenic expression. Overall, these studies define N-domain dependent survival signaling as an Achilles' heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain mediated regulation of bcl-2 family members.

Disclosures:
No relevant conflicts of interest to declare.