ABSTRACT: Bruton Tyrosine Kinase (BTK) is a crucial target for anticancer, anti-inflammatory, and antiviral therapies. BTK inhibitors (BTKis) have revolutionized the therapeutic options for B-cell malignancies, including mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), lymphoplasmacytic lymphoma/Waldenström macroglobulinemia and small lymphocytic lymphoma. Despite the therapeutic efficacy achieved with BTKis, some patients develop relapse and drug resistance. Different new approaches offer an alternative way to prevent or overcome the resistance: by targeting bypass mechanisms by combination therapies;temporal sequencing of therapies;chimeric antigen receptor-modified T (CAR-T) cell therapy;real-time monitoring of patient response; next-generation BTKis (ngBTKis). Above all, ngBTKis and combined treatment are proposed as strategies more effective and less toxic, to improve disease management and patient survival. Epigenomics of B-cell malignancies may explain the intraclonal diversification and help elucidating the reason of emerging relapse and treatment refractoriness/resistance. We identified the first next-generation, selective, reversible, non-covalent BTK-epigenetic inhibitor (BTK-epi-inhibitor) capable of downregulating BTK at both the transcriptional and enzymatic levels.To characterize this molecule, we employed a comprehensive and integrative experimental approach, encompassing genomic, epigenomic, transcriptional, and on-target analyses. We investigated the molecular and functional mechanisms underlying the activity of UVI5008, while also evaluating its therapeutic efficacy, toxicity, pharmacokinetics, and safety profile. These studies were conducted through a combination of in vitro (cell culture), ex vivo, and in vivo (animal model) experiments, aiming to minimize side effects and off-target toxicity. Importantly, we demonstrated that UVI5008 inhibits both wild-type BTK and the C481S mutant isoform—a key mutation associated with resistance to first-generation BTK inhibitors—across in silico, ex vivo, and in vivo models of chronic lymphocytic leukemia (CLL) and B-cell lymphoma. Treatment with UVI5008 led to a significant increase in cell death, accompanied by a reduction in BTK protein levels, its phosphorylation, and downstream signaling activity. Taken together, these data highlight the novelty, potency, and clinical relevance of UVI5008 as a new therapeutic strategy for BTK-driven malignancies, especially in relapsed/refractory (R/R) CLL and B-cell lymphoma, where current therapies often fail.
