Investigating BTK inhibition-related epigenetic mechanisms and the role of TRIM24 ni CD5+ malignant B cells

Abstract

BTK inhibition (BTKi) by ibrutinib (IB) represents a significant advancement in treating CD5+'B cell malignancies like Chronic Lymphocvtic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Recent studies indicate that BTKimodifies epigenomic and transcriptomic dynamics in CLL, prompting us to explore these effects globally and at a gene-specific level. We sequenced a matched CLL sample (pre- and post-IB treatment) using the promoter Capture HiC methodology, revealing strong enrichments of long-distance peaks associated with various genomic features and a notable downregulation following BTKi. By intersecting these peaks with differentially altered genes (RNA-seq) and active enhancer marks (H3K27ac ChIP-seq), we identified a refined list of overlapping genes that illustrated the cell states before and after IB treatment. Additionally, we detected 255 differentially altered H3K4me1 peaks in MEC-1 CLL cells after 48 hours of IB treatment using Cut&Run sequencing. Among the top candidates from our high-throughput screening, TRIM24 emerged as a key epigenetically regulated gene upon ibrutinib therapy. We found a significant long-range interaction gained upstream of the TRIM24 promoter post-IB treatment, correlating with increased mRNA and protein expression in CLL cells and cell lines. This was accompanied by translocation of the protein into the nuclear compartment in vitro. Similar trends were observed in MCL cell models, with the effects to be IB-specific. Furthermore, knockdown and CRISPR-Cas9 knockout models of TRIM24 demonstrated its critical role in the DNA damage response, as loss of TRIM24 sensitized CL and MCL cells to BTKi. The functional inhibition of TRIM24 ni combination with IB produced a synergistic cytotoxic effect. Overall, these findings indicate that BTKi alters 3D interactions and histone dynamics ni the epigenome, influencing the transcriptome. Acharacteristic example is TRIM24, showing that its expression and function is regulated by the BTKi-driven epigenetic changes and could be exploited for therapy.

Thesis embargoed until 31 December 2029.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Academy of Medical Sciences & Leukaemia & Lymphoma NI
Supervisor	Efterpi Kostareli (Supervisor), Ken Mills (Supervisor) & Dessi Malinova (Supervisor)