Genetic and Pharmacologic Targeting of STAT5/Gab2/PI3K/mTOR Signaling in a Mouse Myeloproliferative Disease Model.
Authors: Li Geqiang , Wang Zhengqi , Miskimen Kristy L. , Xie Xiu Yan , Tse William , Gouilleux Fabrice , Moriggl Richard , Bunting Kevin D. 
Published: October 2019

Abstract
Abstract 3902
Poster Board III-838
Signal transducer and activator of transcription-5 (STAT5) is a latent transcription factor that can be activated by phosphorylation by Janus kinases (JAKs) in the cytoplasm, leading to dimerization, DNA binding, and retention within the nucleus. Phosphorylated STAT5 as determined at the single cell level by flow cytometry is a biomarker associated with poor prognosis in certain types of myeloid malignancies including juvenile myelomonocytic leukemia and M4/M5 leukemias. However, targeting of phosphorylated STAT5 or its aberrant signaling might be difficult since direct inhibition of STAT5 transcriptional activity in its entirety may present significant side effects including immune suppression, hematopoietic stem cell dysfunction, or liver problems. Understanding aberrant STAT5 signaling in normal vs. leukemic cells may allow development of novel strategies for leukemia therapy. Our recent demonstrations of cytoplasmic localization of phosphorylated STAT5 in chronic myeloid leukemia and acute myeloid leukemia have lead us to explore to the efficacy of molecularly defining and targeting cooperating signaling pathways. Persistently active STAT5 (STAT5aS711F) has close encounters with Grb2-associated binding protein (Gab2) in the cytoplasm which is associated with increased activation of Akt. Enhanced sensitivity to inhibition of STAT5, SHP-2, and Gab2 has also been described in Bcr/Abl induced oncogenic activities. We have shown that STAT5aS711F expression induces myeloid expansion and promotes growth through a Gab2-dependent mechanism as determined in vitro using Gab2 decoy molecules. However, the potential efficacy of targeting Gab2 in vivo in a live animal disease model has not been explored. Here we have retrovirally transduced wild-type or Gab2-/- mouse bone marrow cells with a murine stem cell virus (MSCV)-based retroviral vector expressing STAT5aS711F upstream of an internal ribosomal entry sequence and the enhanced green fluorescent protein (GFP). Transplanted bone marrow cells caused the expected myeloproliferative disease (MPD) phenotypes that were quantified along with mouse survival. Mice reconstituted with transduced bone marrow cells lacking Gab2 had a significantly attenuated accumulation of myeloid Gr-1+/Mac-1+ cells in the peripheral blood (3.4-fold; P=0.014), liver, and spleen and showed improved survival (control 30 days vs. >90 days without Gab2). However, phosphorylation of Akt activated by STAT5aS711F also occurred in mice lacking Gab2, indicating that Gab2-independent activation of Akt also occurs. Like reported by others in Jak2 and Bcr/Abl mouse MPD models we observed cell extrinsic MPD suggesting that STAT5 activates a paracrine signaling pathway that does not depend on direct Gab2 interaction. This lead us to consider alternative approaches for targeting the PI3K/Akt pathway and to begin defining Akt target genes of importance in STAT5 provoked MPD. Rapamycin is an effective inhibitor of mTORC1 and has been previously shown to synergize with protein tyrosine kinase inhibitors. Therefore, we utilized rapamycin to test whether targeting PI3K/Akt/mTOR signaling would be sufficient to impair cytokine-independent growth provoked by STAT5aS711F. First, BaF3 cells expressing STAT5aS711F were tested for their responsiveness to rapamycin (0.01 – 1 nM). Treatment with rapamycin effectively stopped the cells from growing but was not cytotoxic. Due to the effects of rapamycin on hematopoietic engraftment, it was necessary to optimize transplant and disease burden for rapamycin treatment in vivo. Strikingly, daily treatment of transplanted mice (vehicle N=9; rapamycin N=10) with 4 mg/kg of rapamycin at the early stage of MPD provided a 3.3-fold reduction in Gr-1+/Mac-1+ cells (P=0.002) and prolonged survival of treated mice (>90 days with rapamycin compared with 60 days for control). This effect was cytostatic but did not prevent the subsequent recurrence of MPD once the treatment was stopped. The use of additional drugs and/or optimized transplant/treatment protocols will be required for future studies Overall, these data provide in vivo evidence for clinically significant cooperative signaling by STAT5 and AKT-mTOR pathway in leukemic hematopoiesis and support targeting of the Akt-mTOR axis as therapy for MPDs with Jak2 or Mpl mutations and phosphorylated STAT5.

Disclosures:
No relevant conflicts of interest to declare.