Mechanism of Negative Feedback Regulation of Oncogenic BCR-Signaling in Mature B-Cell Lymphoma

Background and Significance: CD25 (IL2RA) has been extensively studied as IL2-receptor chains expressed on T- and NK-cells. However, CD25 is sharply upregulated upon BCR-signaling. Mature B-cell lymphoma subtypes (CLL, MCL, DLBCL) that depend on oncogenic BCR-signaling inactivate CD25 by proteolytic cleavage and aggressiveness of disease is associated with low expression CD25 surface levels and high serum levels of cleaved CD25.

Results: Here, we discovered a mechanistic framework of dynamic BCR-feedback control and its dependency on coordinated activity of PKCδ and CD25. To study the role of CD25 in a model of aggressive B-cell lymphoma, we propagated splenic B-cells from CD25-fl/fl mice and transduced them with the CARD11-L232LI oncogene. Induction of Cre-activity in this model resulted in rapid loss of CD25 surface expression and dramatic acceleration of proliferation of CD25-/- B-cell lymphoma cells. Conditional ablation of CD25 during normal B-cell development in vivo (Cd25-fl/fl x Mb1-Cre) resulted in the development of B-cell autoimmunity with pervasive autoantibody production, development of spontaneous germinal centers. RNA-seq and mass spectrometry-based global phosphoproteomic studies revealed that acute ablation of CD25 in CARD11-L232LI B-cell lymphoma resulted in prominent activation of NF-kB gene expression programs, hyperphosphorylation of multiple substrates in the BCR-signaling pathway, including SYK, Src-family kinases, BLNK, NF-kB-components and PKCδ. Conversely, CD25-deletion caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Imbalances of control of BCR-signalling following CD25-deletion were confirmed by Western blot, namely hyperphosphorylation of SYK BLNK, ERK, NFKBIA and PKCδ as well as loss of SHP1- and SHIP1-phosphorylation. In addition to Cre-mediated deletion of CD25 in murine B-cell lymphoma cells, we confirmed these results by CRISPR-mediated deletion of CD25 in patient-derived mantle cell lymphoma xenografts (PDX).

Mechanistic studies revealed that oncogenic BCR signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail (S268, T271). Genetic deletion of PKCδ and mutations of CD25 S268/T271 residues demonstrated that PKCδ-mediated CD25-phosphorylation was critical for CD25-dependent feedback control of oncogenic BCR signaling. Our genetic and interactome studies revealed that BCR-signaling induced PKCδ-mediated phosphorylation and recruitment of CD25 to the BCR. Instead of transducing IL2-signals, CD25 directed ITIM-mediated SHP1-activation towards BCR-signaling molecules, within range of SHP1-mediated dephosphorylation. In proximity of the BCR, CD25 attracted ITIM-receptor nanoclusters, resulting in local concentration and activation of SHP1 for negative feedback control of BCR-signaling. Defective BCR-feedback control in human CD25-/- germinal center B-cells could be restored by a novel bispecific antibody to direct ITIM-dependent SHP1 activation towards BCR-proximal signaling molecules.

BCR-dependent B-cell lymphomas frequently harbor deleterious mutations of PKCδ and CD25 is typically inactivated by proteolytic cleavage. To investigate the interplay between PKCδ and CD25, we introduced genetic CD25 knockin alleles using HDR templates encoding GFP and wildtype CD25 or CD25-S268A/T271V (AV) mutations in primary human germinal center (GC) B-cells cultured on YK6 follicular dendritic cells. Interestingly, CD25-AV mutant 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 the 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.

Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, our findings highlight the previously unrecognized role of PKCδ and CD25 in assembling inhibitory phosphatases to control BCR signaling. Also in the context of oncogenic BCR-signaling in B-cell lymphoma, CD25 and PKCδ cooperate as negative regulators. These findings are consistent with frequent inactivation of CD25 by proteolytic cleavage as well as PKCδ-mutations in B-cell lymphomas.

Hodson:Astra Zeneca, GSK: Research Funding. Marson:Site Tx: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arsenal Biosciences: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellanome: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; NewLimit: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spotlight Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Survey Genomics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Tenaya: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Lightcast: Current holder of stock options in a privately-held company, Research Funding; Gilead: Research Funding; Pfizer: Research Funding; 23 and Me: Research Funding; PACT Pharma: Research Funding; JunoTherapeutics: Research Funding; Trizell: Research Funding; Vertex: Research Funding; Merck: Research Funding; Amgen: Research Funding; Genentech: Research Funding; GLG: Research Funding; ClearView Healthcare: Research Funding; AlphaSights: Research Funding; Rupert Case Management: Research Funding; Bernstein: Research Funding; Alda: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; the Emerson Collective: Research Funding; Arc Institute: Research Funding; Epinomics: Research Funding; Sanofi: Research Funding; Anthem: Research Funding.