139P - Rewiring of cis-regulatory and kinase signalling networks in acalabrutinib-resistant ABC DLBCL cells

Background

Bruton's tyrosine kinase inhibitors (BTKi) like ibrutinib and acalabrutinib are initially effective in Activated B-cell Diffuse Large B-cell Lymphoma (ABC DLBCL), but resistance develops rapidly. While some BTKi-resistant clones show coding mutations in BTK or its immediate effectors, such mutations are not obligatory for resistance. We hypothesized that BTKi resistance is underpinned by rewiring of signalling and/or cis-regulatory control of BTK-responsive genes.

Methods

We generated a polyclonal BTKi-resistant subpopulation of U2932 ABC DLBCL-derived cells by culturing in increasing acalabrutinib concentrations. Exome sequencing confirmed the absence of coding mutations. We profiled gene expression (RNA-seq), chromatin state (ATAC-seq), promoter-enhancer contacts (Promoter Capture Hi-C), and kinase signalling (targeted phosphoproteomics) in BTKi-naive and resistant cells. Results were integrated using gene regulatory and kinase network analyses. Additionally, we identified BTK-response genes and enhancers in healthy human B-cells activated with and without acalabrutinib.

Results

Acalabrutinib-resistant cells acquired a distinct gene expression profile maintained in the presence and upon short-term withdrawal of acalabrutinib. Most BTK-response genes remained derepressed in resistant cells. We observed changes in accessibility of only a small number of enhancers, suggesting maintained activity by upstream signalling. Kinase signalling analysis revealed altered cascades downstream of B-cell receptor in resistant cells.

Conclusions

Acquired acalabrutinib resistance can be underpinned by rewiring of the B-cell receptor signalling axis without coding mutations or large-scale changes in chromatin accessibility. Ongoing analyses focus on identifying potential driver events of altered signalling.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

Medical Research Council, London Institute of Medical Sciences, Imperial College London.

Funding

Medical Research Council.

Disclosure

M. Spivakov: Financial Interests, Personal, Stocks/Shares: Enhanc3D Genomics. All other authors have declared no conflicts of interest.