B-cell receptor and Toll-like receptor signaling in Chronic Lymphocytic Leukemia (CLL) proliferation

The B-cell receptor (BCR) pathway plays a key role in the pathogenesis of Chronic Lymphocytic Leukemia (CLL) and has emerged as a major therapeutic target in this disease. However, BCR stimulation is not sufficient to induce CLL cell proliferation in vitro suggesting that additional signals from the microenvironment are required to drive the expansion of the malignant cells. Numerous microenvironmental signals have been identified that can increase the survival and/or induce CLL cell proliferation in vitro. These signals typically represent various secreted or cell surface ligands that are expressed by different cell types present in the lymph node tumor microenvironment, such as T cells, macrophages and stromal cells, or molecules that would be expected to be released by apoptotic cells, such as apoptosis associated autoantigens or CpG-unmethylated mitochondrial DNA. Currently, there is considerable evidence that activated CD4 T cells play an important role in regulating CLL cell proliferation, as such cells are often seen in direct contact with CLL cells in lymph node proliferation centers and are required for the expansion of xenografted human CLL cells in immunodeficient NSG mice. Regarding other microenvironmental signals, the extent to which they contribute to the growth and survival of the leukemic cells in vivo has still not been fully established. The goal of this study was to investigate the relevance of some of these microenvironmental signals in regulating the growth and proliferation of the malignant B cells in vivo, with particular focus on BCR- and TLR-derived signals. This question was addressed using two mouse models of CLL and genetic disruption of the BCR- and TLR- signaling pathway by CRISPR/Cas9. The data presented in this thesis show that BCR signals are directly involved in driving CLL cell proliferation and cooperate with genetic lesions in negative cell cycle regulators during Richter transformation. In contrast, we show that cells with knockout of the TLR-signaling molecules IRAK4 or MyD88 are not negatively selected in vivo, arguing against an essential role for TLR-signals in driving leukemia cell proliferation. Finally, we provide evidence for a major role for macrophages in supporting the growth and survival of Richter Syndrome cells. The novel CRISPR/Cas9 genome editing approach that was developed and described in this thesis also represents an important tool to investigate the relative relevance of additional microenvironmental signals for the growth of the malignant cells in vivo, because it allows for rapid disruption of signaling pathways implicated in the proliferation and survival of the leukemia cells.