The role of RhoA in diffuse large B-cell lymphoma

Abstract

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. DLBCL is divided into two major transcriptomic subgroups, activated B-cell-like (ABC) and germinal center B-cell-like (GCB) lymphomas, and further categorized into genetic subtypes based on genetic alterations. DLBCL is genetically heterogeneous, and genetic alterations occur in different types of genes involved in B-cell receptor (BCR) signaling, regulation of transcription and translation, and other pathways controlling cell survival. One such recurrently mutated gene is the cytoskeleton regulator Ras Homolog Family Member A (RHOA). RhoA has been well described for its role in regulating actin polymerization and various signaling pathways in other cell types. Objectives: Therefore, the objective of this work was to unravel the mechanistic role of RhoA and RhoA mutations in DLBCL and additionally elucidate oncogenic survival pathways affected by RhoA mutations. Furthermore, we aimed to evaluate its effects on BCR internalization, the BCR micro-clustering on the cell membrane, the actin network, and lastly to identify optimal therapies for RhoA-mutated DLBCL. Methods: To achieve the objectives and get a complete understanding of the role of RhoA and how this role alters when mutated, we utilized inhibitors, CRISPR synergy/resistance screening, quantitative proteomics, proximity ligation assays, and BioID-based interactome profiling. Additionally, this mechanistic investigation employed the use of endogenous knock-in cells expressing mutant variants of RhoA, inducible knockouts and knockdowns, and RNA sequencing, along with super-resolution microscopy and other molecular, cell biological and biochemical techniques. Results: We uncovered that RhoA plays a critical role in regulating oncogenic BCR signaling and cell survival. It impacted the chronic active BCR signaling pathway in ABC-DLBCL cells by controlling BCR microcluster formation at the cell membrane through rewiring of the actin filament network, which is essential for BCR signal transduction. Thus, loss of RhoA inhibited BCR-proximal signaling and endocytosis of the BCR and subsequently led to the disruption of the My-T-BCR complex and NF-kB activity, resulting in cell death. In contrast, investigation into the recurrent mutations revealed that the R5W mutation specifically results in increased activation of RhoA, leading to enhanced BCR clustering and consequently augmented NF-kB survival signals and reduced responsiveness of ABC-DLBCL cells to Bruton's tyrosine kinase inhibitors. Conclusions: We have shown that RhoA plays an intricate role in controlling BCR microcluster formation and oncogenic BCR signaling. Mutated RhoA, specifically R5W, confers a survival phenotype through specific alterations to the actin cytoskeleton structure resulting in enhanced BCR microcluster formation and signaling. Thus, we have uncovered that mutations in cytoskeletal regulators contribute to intracellular DLBCL survival programs with implications for targeted therapy approaches.