The proposed project is based on the premise that major effects of novel kinase inhibitors involve targets in cells of the CLL microenvironment rather than in leukemic cells. Experiments using Lyn knock out mice, or chemical, specific inhibition of Btk mutants, uniformly show a relatively modest contribution of kinase inactivation in the direct killing or for the apoptosis of malignant B cells, both for Lyn and Btk. In stark contrast, inhibition of both kinases in microenvironmental cells, in particular in macrophages, seems to strongly inhibit CLL growth and enhance apoptosis of CLL cells. This novel concept is corroborated by the experiments in vitro and in vivo. These findings provide an explanation for the lack of direct cell killing effects of both Btk and PI3K inhibitors observed in CLL patients, where leukemic cells are redistributed from lymphoid organs to the peripheral blood for months during treatment. Accordingly, the initial increase of leukemic cell counts in the peripheral blood during inhibitor treatment is a hallmark of these drugs. Taken together, there is cumulating evidence that indirect (microenvironmental) effects rather than direct effects account for the therapeutic benefit of this class of agents.
The precise mechanisms of action of these effects of novel inhibitors are not completely understood. In particular, the therapeutic mechanisms of kinase inhibitors in high-risk CLL, with a high degree of genomic instability (complex karyotype) and the frequent dysfunction of the DNA damage response (DDR) (TP53 dysfunction), are poorly understood. The clinical efficacy of these inhibitors suggests that high-risk CLL cells remain dependent on microenvironmental support to a certain extent, and that inhibitors such as ibrutinib or idelalisib can modify the microenvironment to induce CLL apoptosis despite the absence of functional DDR signaling. In preliminary experiments, we have seen a functional link between Lyn kinase and an enhanced DDR in CLL.
Based on these observations, the overall aim of the second funding period is to clarify on a molecular level the role of kinases (Lyn, Btk) that seem essential for CLL growth in high-risk CLL. To this end, we will explore the interactions of these tyrosine kinases and the DDR in CLL cells. Moreover, we will clarify the role of these kinases for the intercellular dialogue between CLL cells and the microenvironment in high-risk, TP53 dysfunctional CLL. In four specific aims we will address the following questions: 1. Investigate Lyn-mediated support from microenvironmental cells to the development of high-risk CLL. 2. Investigate the autonomous Lyn-mediated modulation of the DDR in CLL cells. 3. Interrogate Btk-mediated microenvironmental interactions in DDR-defective CLL cells with the use of chemico-genetic tools. 4. Test the clonal evolution of high-risk (del(17p), complex karyotype) CLL under treatment with pathway targeted inhibitors. These experiments will not only provide essential insights into the mechanisms of action of these kinases and their inhibitors in CLL, but also provide novel targets for therapeutic intervention.