Abstract A009: PKCδ-mediated feedback control of B-cell receptor signaling and its subversion in diffuse large B-cell lymphoma
Ruifeng Sun, Jaewoong Lee, Kathrin Kläsener, Mark E. Robinson, Kohei Kume, Zhangliang Cheng, Kadriye N. Cosgun, Etienne Leveille, Longhui Zeng, Xiaolei Su, Ning Ma, Nagarajan Vaidehi, Huimin Geng, Eric Meffre, Daniel J. Hodson, Mathhew S. Davids, Michael Reth, Markus MüschenAbstract
Background and Significance:
Diffuse large B-cell lymphoma, activated-B-cell-like type (ABC-DLBCL) is marked by chronic BCR and NF-κB signaling, which are often the result of activating mutations of CD79B, MYD88 and CARD11. In addition, deleterious mutations in negative regulators of BCR signaling, including PKCδ, SHP1 and SHIP, are found in 5-10% of DLBCL. PKCδ and the phosphatases SHP1 and SHIP1 curb excessive B-cell activation and defects in these molecules result in autoimmunity.
Results:
Here, we discovered a mechanistic framework of dynamic BCR-feedback control in activated and germinal center (GC) B-cells that is frequently inactivated in DLBCL. This feedback control depends on coordinated activity of PKCδ, the inhibitory phosphatases SHP1 and SHIP1, and the surface receptor CD25 in assembling inhibitory complexes in proximity of BCR. Proteomic analysis revealed strong induction of PKCδ and CD25 expression upon BCR engagement. Genetic engineering of murine and human B cells identified PKCδ and CD25 as negative regulators of BCR and NF-κB signaling. RNA-seq and phosphoproteomic studies revealed that ablation of PKCδ or CD25 in B cells resulted in prominent activation of NF-kB gene expression programs and hyperphosphorylation of multiple substrates in the BCR-signaling pathway. Conversely, both PKCδ- and CD25-ablation caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Mechanistic studies showed that oncogenic BCR signaling induced PKCd-mediated CD25-phosphorylation on its cytoplasmic tail (S268, T271). To understand the functional importance of PKCδ-mediated CD25 phosphorylation, we introduced genetic CD25 knockin alleles using HDR templates encoding wildtype CD25 or CD25-S268A/T271V (AV) mutations in human GC B-cells. Interestingly, CD25-AV knockin GC B-cells failed to terminate BCR-signaling and showed autonomous Ca2+ oscillations. In addition, CD25-AV knockin GC B-cells expressed increased levels of activation markers CD69, CD80 and CD86, and exhibited constitutive activation of the NF-kB pathway. In coculture experiments determining how CD25 regulates competitive fitness, CD25-AV knockin GC B-cells rapidly outcompeted their CD25-wildtype counterparts. Interactome studies and molecular dynamics simulations revealed that PKCδ-dependent phosphorylation of CD25 was required to retain inhibitory phosphatases in proximity of BCR signaling complexes to attenuate BCR signaling. Moreover, defective PKCδ-induced assembly of inhibitory complexes was restored with a newly developed bispecific antibody and chemically induced heterodimerization that directed SHP1 towards the BCR.
Conclusion:
DLBCLs frequently harbor activating mutations that sustain oncogenic BCR signaling as well as deleterious mutations of inhibitory phosphatases. Our findings show that PKCδ-mediated CD25 phosphorylation is required for assembly of inhibitory complexes in the proximity of BCR, which mediates constitutive BCR signaling suppression in GC B cells but is inactivated in DLBCL to enable unconstrained signaling activity and proliferative advantage.
Citation Format:
Ruifeng Sun, Jaewoong Lee, Kathrin Kläsener, Mark E. Robinson, Kohei Kume, Zhangliang Cheng, Kadriye N. Cosgun, Etienne Leveille, Longhui Zeng, Xiaolei Su, Ning Ma, Nagarajan Vaidehi, Huimin Geng, Eric Meffre, Daniel J. Hodson, Mathhew S. Davids, Michael Reth, Markus Müschen. PKCδ-mediated feedback control of B-cell receptor signaling and its subversion in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A009.