Integrin-linked kinase in normal and malignant B lymphocytes

The fate of mature B cells is predisposed by their interactions with the lymphoid microenvironment. Adhesion molecules and downstream adaptors facilitate the long-distance trafficking of B cells to the lymphoid organs and their appropriate localization therein, allowing them to receive signals for activation and proliferation. Malignant B cells such as chronic lymphocytic leukemia (CLL) cells can utilize a similar adhesive repertoire as normal B lymphocytes but, in adaptation to the tumor microenvironment, also display alterations in adhesion molecule expression and signaling. In CLL, the lymphoid microenvironment is a major contributor to the development of chemoresistance and relapses. We have previously identified the beta1 integrin VLA-4 as a key molecule in CLL cell homing, bone marrow infiltration with leukemic cells, proliferation, and prediction of clinical outcome. Integrin-linked kinase (ILK) is an adaptor protein that links beta1 integrins to the actin cytoskeleton and to a range of signalling pathways, with increased levels in various cancers. The contribution of ILK to beta1 integrin function of normal and malignant B cells has not been elucidated yet and is subject of this proposal. In preparatory work, we have observed an overexpression of ILK in VLA-4 positive human CLL samples and a further upregulation of ILK expression in CLL cell activation preceding their proliferation, in an NF-kappaB-dependent manner. In addition, we have established a conditional knockout mouse model with ILK deficiency in the CD19-lineage and have further crossed this model to the TCL1 transgenic murine model for CLL. On basis of the established mouse models and our large collection of primary human CLL samples, we aim to 1) investigate the contribution of ILK to beta1 integrin (particularly VLA-4) function in motility, extravasation, survival, and differentiation of normal B cells, and 2) to dissect its contribution in a malignant context, using chronic lymphocytic leukemia as a model for malignant B cells. 3) We will extend and validate our results using primary human CLL cells. We will test whether ILK is needed for integrating signals inducing motility/homing and/or cell cycle progression/retention of the tumor cells; and we will dissect potential differences in ILK usage of normal and malignant B lymphocytes. Our findings will aid to evaluate beta1 integrin signals as therapeutic targets in B cell malignancies and other tumor entities with lymphoid involvement.

The fate of mature B cells is determined by their interactions with the lymphoid microenvironment. Adhesion molecules and downstream adaptors facilitate the long-distance trafficking of B cells to the lymphoid organs and their appropriate localization therein, allowing them to receive signals for activation and proliferation. Malignant B cells such as chronic lymphocytic leukemia (CLL) cells can utilize a similar adhesive repertoire as normal B lymphocytes but, in adaptation to the tumor microenvironment, also display alterations in adhesion molecule expression and signaling. In CLL, the lymphoid microenvironment is a major contributor to the development of chemoresistance and relapses. We have previously identified the beta1 integrin VLA-4 as a key molecule in CLL cell homing, bone marrow infiltration, proliferation, and prediction of clinical outcome. Integrin-linked kinase (ILK) is an adaptor protein that links beta1 integrins to the actin cytoskeleton and to a range of signaling pathways, with increased ILK levels observed in various cancers. The contribution of ILK to beta1 integrin function and development of normal and malignant B cells was subject of this proposal. To this end, we have established a conditional knockout mouse model with ILK deficiency in the CD19-lineage and have further crossed this model to the TCL1 transgenic murine model for CLL. Major aims were to investigate the contribution of ILK to beta1 integrin (particularly VLA-4) function in motility, extravasation, survival, and differentiation of normal B cells, and to dissect its contribution in a malignant context, using chronic lymphocytic leukemia as a model for malignant B cells. We achieved following new results: 1. ILK is dispensable for the development of normal B cells. 2. ILK deficiency does not affect migration, adhesion or proliferation of normal B cells. 3. B cell specific genetic ablation of ILK in the Tcl1 transgenic mouse model of CLL results in decelerated leukemia development. 4. Decelerated leukemia development in absence of ILK is primarily based on reduced organ infiltration and proliferation of CLL cells. 5. In CLL patient samples, ILK expression is increased in association with markers of poor prognosis, in recently divided cells, and in proliferative lymphoid areas. 6. ILK expression is upregulated in activating co-cultures of CLL and T cell and correlates with induction of CLL cyclin D1. 7. In CLL, ILK expression is regulated by NF- B signaling, which is triggered by TNF-a. 8. ILK localizes to centrosomal structures and is required for centrosome clustering during proliferation of leukemia cells.