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Investigation of the role of CYLD in Chronic Lymphocytic Leukaemia

Periodic Reporting for period 1 - CIRCLE (Investigation of the role of CYLD in Chronic Lymphocytic Leukaemia)

Reporting period: 2016-04-15 to 2018-04-14

Chronic Lymphocytic Leukemia (CLL) is the most common type of Leukemia in the western world, affecting 6 every 100,000 individuals every year and is characterized by the accumulation of CD19+/CD5+ B cells in the peripheral blood and lymphoid organs of the patients. It is considered a disease of the elder male, since the median age at diagnosis is 72 years and the proportion of male:female patients is 2:1. CLL has a median survival of 10 years but the course can be very heterogeneous. In some more aggressive cases, patients succumb to the disease after 2-3 years, whereas in indolent cases the lifespan of the patient may not be affected. The cause of the disease is unknown, even though disease susceptibility and prognosis has been associated with several genetic factors. CYLD is a functional ubiquitinase, involved in the removal of ubiquitin chains that were attached to proteins to change their structure and enable interaction with other proteins in signaling pathways, rather than proteasomal degradation. CYLD was discovered in patients affected by a benign form of skin tumor but it was later revealed to have a tumor suppressor action in different types of solid and hematological tumors.
Despite introduction of novel therapeutic agents, CLL remains at large incurable generating an important societal burden, both in terms of caring and well-being of the general population and of treatment costs. With the EU population growing constantly older, such diseases are expected to pose an ever-growing risk, and an EU strategic agenda has been implemented to address it.
The overall objective of the project was to elucidate the role of CYLD in CLL, using in vivo and in silico models. Understanding the role of CYLD in CLL could reveal novel targets for therapeutic interventions or markers for disease prognosis.
The project has been developed along 2 lines: in vivo and in silico studies.
For the in vivo studies we first generated three animal models with a targeted deletion of the catalytically active form of CYLD in B lymphocytes. Since abnormal accumulation of CD19+/CD5+ B lymphocytes is the hallmark of CLL, we targeted deletion of the protein in these cells, using the well-established Cre-LoxP system. Generation of B cells is a multi-step, tightly controlled process, which leads to the development of mature fully functional B cells. Using different targeting strategies, we generated two mice lines that lack expression of the catalytically active form of CYLD starting from different B cell maturation stages. In the third animal model, we combined ablation of the catalytically active form of CYLD with a widely-used model of CLL in mice, with the aim to evaluate whether ablation of CYLD activity on B cells would influence CLL progression.
Extensive characterization of the generated mouse lines indicated that the mice had an unexpected, but extremely interesting phenotype. Mice lacking expression of the catalytically active form of CYLD in B cells from the initial stages of the maturation process exhibited a striking defect in the maturation of B cells. These mice had virtually no B cells in their peripheral blood, and the populations of fully mature B cells in the bone marrow and the spleen were severely depleted. Macroscopic analysis of the spleen, indicated that the spleen of the transgenic animals was markedly smaller, both in terms of size and cellularity, whereas histopathological examination of the spleen indicated that the microarchitecture and the organization of the organ were deeply affected. From a functional standpoint, mice lacking expression of the catalytically active form of CYLD could not successfully mount an immune response following immunization with commonly used prototype immunogens, thus indicating that these mice were severely immune-compromised. The transgenic mice in which ablation of the catalytically active form of CYLD was combined with a genetic model of CLL also displayed a substantial reduction of the mature B cell compartment. The few mature B cells remaining in these animals did not appear to be more prone to switch to a CLL-like phenotype.
For the in silico studies we succeeded in securing access to a database containing RNA sequencing data from purified B cells of approximately 300 CLL patients. These data are being analyzed to establish CYLD expression patterns and concomitant alterations of the associated signaling pathways. Our preliminary data indicates a downregulation in the expression of CYLD in CLL patients. The extreme depletion of mature B cells in transgenic animals not expressing the catalytically active form of CYLD in B cells has precluded efficient separation of mature B cells, rendering RNA sequencing and further in silico analysis impossible to perform.
The transgenic animal models generated could evolve into immunocompromised models with wide use in disease modeling and drug testing. The results of the study were selected for oral presentation at the 2017 American Society for Hematology (ASH) annual conference, probably the most prestigious conferences of the field with over 30000 attendees.
Our data imply a crucial role for CYLD in the maturation of B cells. Previous studies on the role of CYLD in B cell lymphopoiesis originated from animals with a constitutive knock out of the protein, i.e. the protein was absent in all cells of the organism, and led to controversial results, ranging from no effect to B cell hyperplasia and lymphoid organ enlargement. Our results are based on B cell-specific ablation of the catalytically active form of CYLD, which constitutes a much more refined approach for studying the role of the protein of interest. Transgenic animals with B cell-specific ablation of the catalytically active form of CYLD had a severe impediment in the maturation of B cells, indicating that the catalytic activity of CYLD is of paramount importance for the process.
This finding paves the way for the determination of the role of CYLD, and in a broader sense of functional ubiquitination, in the regulation of various processes, possibly through signaling pathways that had not previously been associated with CYLD and ubiquitination. A deeper understanding of the mechanisms underlying B cell lymphopoiesis may prove beneficial to understand the pathogenesis of many pathological states of B cells. At the same time, unraveling ubiquitin-associated signaling mechanisms could lead to the discovery of novel targets for therapeutic intervention. The long-term socio-economic impact of these findings could be very profound as novel efficient drugs could emerge that would ameliorate the quality of life of the patients and the care-givers, reduce treatment costs and generate revenue for the European pharmaceutical companies.