CD44-hyaluronan interactions in the pathophysiology of CLL

B-cell chronic lymphocytic leukemia (CLL), the most prevalent leukemia in Western societies, is characterized by the progressive accumulation of CD5+ malignant B lymphocytes within the blood, the bone marrow (BM) and the lymph nodes (LNs). The proliferation and survival of CLL cells is highly dependent on their interactions with the tumor microenvironment. While the peripheral blood pool consists of mainly cell cycle arrested CLL cells, the LNs and to a lesser extent also the BM contain specialized proliferative areas, the so-called pseudofollicles. These areas are interspersed with CD4+ CD40L+ subsets of T lymphocytes suggesting that CLL activation by T cells may support the outgrowth of the malignant clone. The adhesion receptor CD44 participates in lymphocyte cell activation, migration and tissue retention via interaction with the extracellular matrix component hyaluronic acid (HA). CD44-HA interactions have been implicated in a variety of tumors. In CLL, elevated CD44 serum levels and CD44 variant (CD44v) isoform expression have been suggested as markers for disease progression but the although the underlying biological mechanisms remain elusive. This proposal aims to dissect how CD44(v) and HA contribute to the appropriate localization of CLL cells in lymphoid proliferation centers and the physiological consequences arising therefrom, with a particular focus on CD40L-mediated regulation of CD44. By using in vivo short-term adoptive transfer assays of human CLL cells to NOD/SCID mice and in vitro shear flow assays, we will dissect the contribution of CD44-HA interactions in CLL cell homing to lymphoid organs. Second, we will study the impact of CD40L-mediated CLL activation on CD44(v)-mediated adhesion on HA substrates and its consequences on survival and proliferation signaling. Finally, the results will be validated in vivo in a murine CLL model with a conditional CD44 knockout in the malignant B cells. Consequences of this CD44 deletion on leukemic dissemination, tumor cell survival and proliferation and on the development of chemoresistance of the animals will be assessed. Collectively, our data will contribute to a better understanding how the microenvironment induces tumor cell proliferation and chemoresistance in CLL via modulation of adhesive interactions. Therapeutic interference with highly affine CD44(v)-HA interactions may release the tumor cells from their protective niches, prevent their proliferation, and eventually provide strong synergism with conventional therapies.

Chronic lymphocytic leukemia (CLL) represents the most prevalent leukemia in Western countries and is characterized by an accumulation of malignant B cells in blood, bone marrow and secondary lymphoid organs. CLL tumor cell proliferation and survival both rely on signals from the tumor microenvironment within lymphoid organs. The increasing knowledge on these processes is reflected by the recent success of novel drugs that act, at least in part, by disrupting leukemia cell- microenvironment interactions. It is therefore of both basic and translational-clinical interest to better understand the molecular mechanisms that result in tumor cell infiltration of protective and proliferation-promoting zones of the lymphoid organs. The CD44 adhesion molecule plays a critical role in the activation, migration and retention of lymphocytes through its strictly regulated interaction with the ligand hyaluronic acid (HA). In addition, the expression of CD44 and in particular of CD44 variants (CD44v) is associated with various cancers. The aim of this project was to determine the contribution of CD44(v)-HA interactions to the localization of the malignant B cells in lymphoid zones and to investigate the pathophysiological consequences. Therefore, a transgenic mouse model for CLL with a B cell-specific CD44 deficiency was established, characterized, and complemented by in vitro tests for migration, activation and proliferation of both, murine and human leukemia cells. In addition, adoptive transfer experiments of human CLL cells in immunodeficient mice were performed. Our analysis led to the following new findings:1. Signals from the tumor microenvironment induce a transcriptional regulation of certain CD44 isoform variants with specific post-translational modifications in CLL cells.2. This switch of the standard to the variant form results in an increased binding to the ligand HA, which is abundantly presented by stromal cells in lymphoid organs. This increased interaction results in a stop signal for the migrating tumor cells, which allows them to settle down in the protective niche and subsequently proliferate.3. A genetic loss of CD44 in B cells of a transgenic leukemia mouse model leads to delayed CLL onset, which is driven by a reduction in tumor cell proliferation in spleen while the bone marrow is of subordinate importance. The spleen microenvironment leads to a transcriptional induction of the variant 6, which is responsible for leukemia cell proliferation, by activating the NF-kB signaling pathway.In summary, we propose a cycle of NF-kB signaling induced by the microenvironment, that leads to CD44 variant 6 expression, and the proliferation of leukemia cells contributing to the pathogenesis of the CLL. Our results contribute to the understanding of molecular mechanisms of tumor pathogenesis in the context of tumor cell-microenvironment and immune cell communication.