Stat5 serine phosphorylation in Bcr/Abl-induced leukemia

Jak-Stat signaling has been implicated in multiple forms of solid as well as hematological cancers. In particular, the constitutive activation of Jak-Stat signaling is found in several forms of leukemia. We have recently identified the transcription factors Stat5a/b as critical nodes in the signaling network downstream of the leukemia-associated Bcr/Abl oncogene. Most importantly, the requirement for Stat5a/b signaling extends to the leukemic stem cell (LSC) compartment. We were able to show that even lowering Stat5a/b levels in already established leukemias inhibits disease and makes Bcr/Abl+ LSCs unable to induce disease upon secondary transplantation. The inhibition of Bcr/Abl+ LSCs is one of the key issues in leukemia therapy as the Bcr/Abl inhibitor Gleevec is not capable of targeting and eliminating these cells. As a consequence, patients are required to take Bcr/Abl kinase inhibitors life- long. There is thus an urgent requirement for new therapeutic strategies to target LSCs. Interestingly, a constitutively active version of Stat5a induces highly aggressive leukemia in mice; this effect is not shared by a constitutively active version of Stat5b. Moreover, we have shown that re-expression of Stat5a restores survival and proliferation of in Stat5a/b - deficient leukemic cells. Hence, we will focus on Stat5a in this proposal. Recently, first insights have been gained into the molecular mechanisms that account for Stat5a-mediated tumor- support. Phosphorylations on serine residues 725 and 779 were found to be critical for the oncogenic function of Stat5a in a murine model of leukemia using the constitutively active version of Stat5a to drive disease. In the proposed study we shall explore the effects of Stat5a serine phosphorylation on homo- and heterodimerization, DNA-binding, Tyr 694 phosphorylation and leukemogenesis. Using well-established murine leukemia models, we intend to investigate whether serine phosphorylation is required to support leukemia induced by the clinically relevant oncogenes Bcr/Ablp185 and Bcr/Ablp210, which drive acute lymphoid leukemia (ALL) and chronic lymphoid leukemia (CML), respectively. Targeting phosphorylation sites by kinase inhibitors is a valid approach to the development of novel therapeutic strategies. We shall also put emphasis on the effects of Stat5a serine phosphorylation within the LSC compartment and identify target genes that depend on serine-phosphorylated Stat5a in the LSC compartment.

JAK-STAT signaling has been implicated in multiple forms of solid as well as hematological cancers. In particular, constitutive activation of JAK-STAT signaling is found in several forms of leukemia. Before we embarked on this project, we had identified STAT5 as a key factor in the initiation and maintenance of BCR/ABL+ leukemia. In these forms of leukemia, STAT5 functions as a critical node in the complex signaling network downstream of the BCR/ABL oncoprotein. In this project we aimed at identifying critical residues within the STAT5 protein that confer its transforming properties. The identification of such residues was supposed to nail down vulnerable and hence therapeutically exploitable sites of STAT5. We examined two prominent sites within the STAT5 protein: so-called serine sites (as positions 725 and 779), which are phosphorylated in tumor cells and render STAT5 active. We explored the effects on BCR/ABL-induced leukemia formation if these sites are lost. To do so, we used mouse models that closely mimic human BCR/ABL+ leukemia. We succeeded and showed that loss of these serine sites blocked the oncogenic function of STAT5 by e.g. prohibiting the nuclear translocation of this transcription factor. If STAT5 does not move to the nucleus, its target genes (responsible for continuous cell proliferation and survival) are not transcribed the tumor cell dies. Hence, the inactivation of STAT5 by blocking its serine phosphorylation represents a novel therapeutic strategy to combat STAT5-dependent diseases, hematopoietic and solid tumors alike. Within the scope of this project we even went one step further and identified the kinases that phosphorylate STAT5 on S725 and S779: CDK8 and PAK1/2, respectively. The blockage of these kinases might represent a constructive way to shut off STAT5 in tumor cells.