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Group Sexl

Jak-Stat involvement in leukemogenesis and NK-mediated tumor surveillance


The focus of the proposed project part is to study Jak-Stat signaling in leukemogenesis and Natural Killer (NK) cell-mediated tumor surveillance. First, we will study mechanisms driving spontaneous leukemogenesis in Stat1-/- animals to understand the tumor suppressing activity of Stat1 in myeloid leukemia. Interestingly, the phenotype of the disease changes upon transplantation and switches from a myeloid leukemia to a blast crisis-like disease displaying B cell-specific surface markers. We will investigate whether this change reflects a certain plasticity of the leukemic cells or whether different leukemic sub-clones drive the disease. Finally, we will identify signaling pathways accounting for leukemogenesis and disease maintenance in the absence of Stat1. Preliminary studies revealed that neither Stat3 nor Stat5 is activated; potential candidate signaling pathways include the NFκB, the hedgehog and the β-catenin pathways.
Recent work, including our own, has taught us that studies on signaling cascades have to consider cell-intrinsic and -extrinsic effects, i.e. the function of a particular signaling pathway in the leukemic cell and in the interaction with the tumor-environment, including immune cells. In leukemia, NK cells represent the main immune cell compartment responsible for tumor clearance. Hence, our second objective relates to Stat1 signaling in NK cell-dependent tumor surveillance. Whereas Stat1-/- NK cells are immature and incapable of efficiently lysing leukemic cells, we did observe a partial rescue in NK cells derived from Stat1Y701F mice that harbor a point mutation in the critical Jak-dependent tyrosine phosphorylation site. This suggests a critical non-canonical function of Stat1 in NK cells. Further support for a central function of Stat1 in NK cell biology stems from our observation that Cdk8-dependent phosphorylation of Stat1S727 inhibits cytotoxicity. Accordingly, NK cells derived from Stat1S727A mice are hyperactive and efficiently protect Stat1S727A mice from tumors under NK cell-surveillance. Notably we observe Stat1 in NK cells at in the immunological synapse upon interaction with tumor target cells.
In the proposed subproject we aim to define both the leukemia-initiating cell and the cancer maintaining or cancer stem cell responsible for disease maintenance and the relevant signaling pathways in the absence of Stat1. Furthermore, we plan to unravel the function(s) of Stat1 within the immunological synapse and to study the impact of Stat1 on NK cell development and maturation.

Goals and Key Hypotheses:

Our aims are to identify the activities of Stat1 that suppress myeloid leukemia and account for NK cell-mediated tumor surveillance.
Working hypotheses for project 1:
Based on the finding that loss of Stat1 is sufficient to induce myeloid leukemia – thereby defining Stat1 as tumor suppressor – we have observed that leukemogenesis occurs in distinct steps and that Stat1-/- leukemic cells show a consistent activation pattern of signaling pathways that maintain the leukemic state. This leads us to propose the following two working hypotheses:
(1) Loss of Stat1 in myeloid precursors - representing the cancer cells of origin (COC) - initiates the disease that requires an additional hit to manifest. Thereafter, disease maintenance is accomplished by a Lin+ leukemic stem cell (LSC).
(2) Stat1 loss triggers and/or complements alterations in other pathways, including NFγB, β-catenin and hedgehog signaling.
Working hypotheses for project 2:
While investigating the role of Stat1 in NK cells we observed that NK cell maturation depends on Stat1, while NK cells derived from Stat1S727A mutant mice display higher cytotoxicity. This leads us to propose the following three working hypotheses:
(1) Stat1-dependent NK-cell maturation requires cell-extrinsic canonical Stat1-dependent MHC class I expression in the bone marrow.
(2) For full-fledged NK cell cytotoxicity, non-canonical cell-intrinsic functions of Stat1 are required in the immunological synapse.
(3) A tyrosine phosphorylation-independent function of Stat1 (U-Stat1) in the immunological synapse is required for NK-cell cytotoxicity.

Our specific goals are as follows:
Project 1:
(1) determine the COC as the origin of the disease; we will exploit Stat1fl/fl mice to delete Stat1 in all hematopoietic cells (vav-Cre), in myeloid progenitors (ltf-Cre) and in B lymphoid (Mb1-Cre) cells.
(2) conduct RNA-Seq studies to determine disease-relevant signaling pathways triggered by loss of Stat1.
Project 2:
(1) analyse maturation of NK cells lacking Stat1 in NK cells only (Stat1fl/fl Ncr1-Cre) and in NK cells only as well as in the entire hematopoietic compartment (Stat1fl/fl vav-Cre).
(2) compare transcription by performing ChIP-Seq in wild-type, Stat1S727A, and Stat1Y701F NK cells.
(3) identify the interaction partners of Stat1 in NK cells using wild-type-, Stat1Y701F- and Stat1S727A-derived NK cells by immunoprecipitation of FLAG-tagged Stat1 followed by mass spectrometry.

Antwort auf/zuklappen Report on funding period 1 (03/2006-03/2010)

Antwort auf/zuklappen Report on funding period 2 (03/2010-03/2013)


Institute of Pharmacology and Toxicology
University of Veterinary Medicine Vienna, VMW
A-1210 Vienna, Austria

phone:  +43(0)1 25077-2910; +43(0)1 664 60257-6291