Aberrant c-AMP Signalling in Richter Syndrome Revealed By Single-Cell Transcriptome and 3D Chromatin Analysis

Background: Richter syndrome (RS) is a severe progression of chronic lymphocytic leukaemia (CLL) into aggressive lymphoma, with a poorly understood molecular basis and an urgent need for new treatments. The application of single-cell RNA sequencing (scRNA-seq) has yielded numerous insights into the genetic events of lymphoma. Within the genome, three-dimensional chromatin structures are dynamic and could play a crucial role in transcriptional regulation.

Methods: We integrated scRNA-seq and high-throughput chromosome conformation capture (Hi-C) sequencing to analyze a patient with CLL that transformed into diffuse large B-cell lymphoma (DLBCL). After RS diagnosis, paired samples of peripheral blood and lymph node tissue were collected.

Results: Overall, 10434 single-cell transcriptomes passed quality control and three major cell types were identified: B cells, cytotoxic T cells and haematopoietic precursor cells. Six clusters of B cells were identified. According to the pseudotime trajectories and the expression level of CD20 in B cells, CLL cells were mostly mapped to Clusters 0, 2, 4 and 6; RS B cells were mostly mapped to Cluster 1. GO analysis revealed that the differentially expressed genes (DEGs) were enriched in immune system processes. B cells undergo dynamic genome architecture reorganization during differentiation and neoplastic transformation. Hi-C sequencing revealed that the interaction matrices of DLBCL cells exhibited enhanced proximal interactions and weakened distal interactions compared with those of CLL cells. DLBCL cells had increased intrachromosomal contact probabilities at <50 Mb and decreased at >50 Mb, with proximal contacts significantly exceeding distal ones. Genomic regions can be assigned into the active compartment (compartment A) and the inactive compartment (compartment B). At the compartment level, DLBCL cells showed strengthened interaction between B compartments (StableBB) and compromised StableAA and SwitchAB. At the topologically associating domain (TAD) scale, the insulation score increased in the DLBCL cells, with increased intra-TAD and decreased inter-TAD contacts, indicating that the TADs were compacted and that the interactions between adjacent TADs were inhibited. Aggregate peak analysis also revealed differential interactions between DLBCL cells and CLL cells. We observed that 6.4% of the DEGs, including 7.0% of the upregulated genes and 5.4% of the downregulated genes, were located at the TAD boundaries. The upregulated genes inside merged TADs (UPGs^TAD) were involved in wound healing, cAMP-mediated signalling and proteasomal processes. KEGG analysis revealed that the UPGs^TAD were enriched in exosome and apoptosis signalling, whereas the DWGs^TAD were enriched in ribosome signalling. ATF1 and CAP1 are representative UPGs^TAD involved in cAMP-mediated signalling.

Conclusion: Using longitudinal samples, we conducted a combined scRNA-seq and Hi-C analysis, demonstrating that RS lymphoma cells exhibited enhanced proximal interactions between chromosomes and reorganized chromatin, thus affecting cAMP-mediated signalling, which has been described to be relevant for a wide array of intracellular processes. Our study is the first to explore the chromatin-reorganization-gene regulation link in RS. We propose that it is worthwhile to identify how the fusion of TADs in transformed DLBCL cells results in transcriptome alterations, highlighting the need for further investigation into cAMP signalling and its role in RS.