''From Molecular Resistance to Tumor Evolution: Insights in BTK Inhibitors, Tumor Microenvironment and Genetic Variants.''

Bruton’s tyrosine kinase (BTK) prevails as a key mediator in the elaborate system of immune signaling, with its essential role in maintaining homeostasis of the B lymphocyte compartment. More specifically, BTK emerges as an anchor target in the advancement of precision medicine across conditions like the inherited immunodeficiency X-linked agammaglobulinemia (XLA) as well as hematological malignancies. In XLA, variations in the gene encoding BTK lead to a profound primary immunodeficiency in boys, marked by the near-total absence of B lymphocytic cells. While acquired, constitutively active BTK mutants have not been detected in B-cell malignancies, studies have emphasized the critical role of BTK-dependent pathways in the development and progression of these pathologies. The duality of BTK as both a critical immune regulator and a therapeutic target stresses its importance in disease biology. From the initial description of XLA in the literature to the cloning of BTK, the subsequent development of the first efficient BTK inhibitor (BTKi), Ibrutinib, in 2007 marked a significant milestone. This achievement led to its rapid approval in both the United States and Europe for the treatment of Chronic Lymphocytic Leukemia, Mantle Cell Lymphoma, and Waldenström’s Macroglobulinemia. However, a new challenge emerged: resistance to Ibrutinib, mainly driven by the acquisition of resistance mutations in the BTK gene. This led to the development of second- and third-generation BTKi, designed to offer enhanced specificity with reduced side effects and with higher dosage potential. Building on the concepts outlined above, this thesis seeks to advance the understanding of BTK by exploring its role through a series of interconnected projects: evaluating the impact of BTK mutations on resistance to targeted inhibitors in vitro, investigating the effects of BTKi in an animal model, TCL1 chronic lymphocytic leukemia cells and their tumor microenvironment (TME), as well as exploring the wider implications of synonymous mutations within the human BTK gene and other critical genes. To achieve these objectives, the first project utilizes a cellular model based on the hematopoietic K562 cell line, carrying an engineered BTK loss-of-function mutation. The study plasmids were generated after thoroughly reviewing the literature, particularly the studies by Wang et al. (2022) and Naeem et al. (2023). This was to gain a deeper understanding of the resistance and probable sensitivity patterns specific to the reversible BTKi, Pirtobrutinib, by comparing it with the irreversible binders, Ibrutinib and Zanubrutinib. Through electroporation K562 cells were transfected with the mutants, and evaluation of activation-dependent phosphorylations was by Western Blot analyses. The second project utilizes a BTKi-resistant C481S mutation/TCL1 transgenic mouse model which allows for the generation of tumor cells that are resistant to BTKi treatment in a TME which is not. Previous studies of plasma biomarkers from patients with chronic lymphocytic leukemia (CLL) treated with BTKi have demonstrated that most changes are derived from the TME. The goal was to investigate potential treatment impact of BTKi on the TME. The third project primarily focused on utilizing three major databases—LOVD3, GnomAD, and All of Us—to create a comprehensive new database of synonymous mutations in the human BTK gene. This concept was later expanded to include other disease-related genes, such as F8 and F9, for comparison with BTK, and eventually extended to include genes like BRCA1, BRCA2, and MH1, among others. The ultimate goal of this project is to use various bioinformatic and statistical analyses to establish a direct link between synonymous mutations and specific pathologies, addressing the fact that these mutations are often overlooked in clinical settings, potentially delaying diagnoses or affecting appropriate treatment.