Exploring the Role of Targeting the PH Domain in BTK-Mediated Genes Expression within Mantle Cell Lymphoma Cells

Abstract

Mantle Cell Lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma with a poor prognosis, driven by dysregulated signalling pathways involving Bruton’s tyrosine kinase (BTK). Therapies targeting the BTK kinase domain, such as ibrutinib and acalabrutinib, have transformed MCL treatments introducing a new paradigm of therapies. However, resistance to these therapies often emerges due to mutations within BTK, such as C481S mutation, limiting their current therapeutic efficacy. Addressing key challenges of cancer’s hallmark traits including drug resistance and the adaptive restructuring of cancer cells, remains essential for improving therapeutic outcomes in the clinic for blood cancer patients.

BTK trafficking to the membrane mediated by the pleckstrin homology (PH) domain is crucial for its interaction with other plasmamembrane signalling molecules. A truncated BTK lacking the PH domain (BTK-ΔPH) provides a unique opportunity to investigate how BTK membrane translocation affects MCL cellular behaviour. Advances in genomic research have disclosed the complexity of the MCL genomic landscape, identifying diverse genetic alterations involved in its pathogenesis and progression.

This thesis aims to explore the role of BTK-ΔPH in MCL cells architecture, hypothesising its influence on BTK-mediated gene expression and signalling. Using CRISPR/Cas9 gene editing, BTK variants with truncated PH domains (BTK-ΔPH) were generated in the classical MCL cell line, MAVER-1, inducing a 397 bp truncation in exon 1 of BTK, thus removing its PH domain. Functional assays (trypan blue exclusion and flow cytometrical cell cycle analysis) demonstrated that the BTK-PH domain is crucial for maintaining normal cell proliferation and cycle progression in MAVER-1 cells. The BTK-ΔPH variant showed a marked reduction in proliferative capacity and alteration of phases of the cell cycle compared to wild-type BTK. However, the baseline analysis via Western blotting revealed no considerable changes in BTK expression levels or its downstream kinases. This suggests that the functional effects of BTK-ΔPH are likely driven not by changes in protein abundance, but by altered subcellular localisation, disrupted downstream signalling, or impaired protein-protein interactions. Transcriptomic analysis using RNA sequencing identified 194 differentially expressed genes (DEGs), comprising 137 downregulated and 57 upregulated genes. While BTK showed no observable change in expression, CCND1 (cyclin D1), a key driver of MCL pathogenesis, was among the top five most downregulated genes. This pattern was further validated by qPCR, which also confirmed reduction in CCND1 expression levels in the mutated cells. In addition, enrichment analyses using ORA and GSEA determined altered pathways including reactive oxygen species (ROS) and TNF-α signalling via NF-κB transcription factor. A clustering heatmap of the log2-scaled gene expression levels created two distinct clusters corresponding to the BTK-ΔPH and BTK-WT sample groups across four biological replicates. These pathways potentially modulated lipid metabolism, particularly within glycerophospholipid metabolism pathway, and traditional BTK associated pathways, such as BCR and chemokine receptor signalling, and toll-like receptor (TLR) signalling. Notably, key MCL-related genes also formed distinct clusters, although only CCND1 and SOX11 were differentially expressed. Overall, these outcomes broaden the affected transcriptional networks, suggesting that the BTK- PH domain selectively influences signalling molecules, while its impact on the key genes associated with MCL pathogenesis may be limited.

Network analyses using Gene MANIA and CytoHubba plugins revealed an interconnected DEGs network with 212 nodes and 3,548 edges. Co-expression was the primary interaction type, indicating strong transcriptional co-regulation among these genes. KEGG enrichment analysis highlighted the network’s relevance to pathways such as “FoxO”, “JAK-STAT”, and “HIF-1 signalling” and cancer-related pathways. Key hub genes, such as IL7R, and CAV1, LEF1 were predicted as potential drivers of aggressive behaviour and poor prognosis in MCL. Further regulatory analysis suggested transcription factors (e.g., SP1 and TP53) and miRNAs (e.g., hsa-miR-34a-5p and hsa-miR-155-5p) may modulate these DEGs.

Integrating DEGs with The Cancer Genome Atlas (TCGA) data predicted the functional relevance of BTK-PH-specific gene signatures in oncogenesis. Analysis using GEPIA2 and GSCA platform linked these signatures to poor survival outcomes and the activity of ten key pathways, including epithelial-to-mesenchymal transition (EMT) and cell cycle across various cancers. Notably, BTK-PH activity appeared to promote apoptosis, suggesting a paradoxical role in cancer progression by facilitating immune evasion and therapeutic resistance.

This thesis highlights the nuanced role of the BTK-PH domain in MCL biology and its broader implications across cancer types. The truncation may shape cell’s role in the tumour microenvironment, influencing metabolic and immune responses. The combinatorial approaches of computational analyses predict that targeting BTK-PH-dependent mechanisms could serve as potential therapeutic strategies and prognostic biomarkers to overcome drug resistance and improve precision medicine approaches. Experimental validation is essential to confirm to these potential outcomes