Meffre, Eric

Background: Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through b- and g-, but not a-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Results: Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds downstream signaling molecules of the B-cell receptor (BCR). Through these interactions, CD25 mediates negative feedback to BCR signaling in response to antigen-encounter in normal B-cells. Defects in CD25-/-B-cells were not replicated in mice that express CD25 but lack expression of the IL2 cytokine. These findings demonstrate IL2-independent functions of CD25 in B-cells and B-cell derived leukemia and lymphoma. To comprehensively study the interactome of the short cytoplasmic tail of CD25, we performed proximity-dependent biotin identification (BioID). This analysis revealed that the CD25 tail exerts negative feedback control through recruitment of the PKCβ-scaffold RACK1 and the inhibitory phosphatase SHIP1 (see schematic, left). Interestingly, the cytoplasmic tail of CD25 harbors a PKCβ-substrate motif and mutation of a central serine residue (S268) to A268 compromised interactions with PKCβ, its scaffold RACK1 and SHIP1, demonstrating that feedback control was dependent on PKCβ-mediated phosphorylation of CD25-S268. A genetic observation in a family with monogenic autoimmunity confirmed the functional importance of the cytoplasmic CD25-tail motif: a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268. In vitro kinase assay with 62 candidate kinases against recombinant cytoplasmic tail of CD25-S268 or -A268 identified PKCβ as top-ranking kinase hit for CD25-S268 but not CD25-A268. Our genetic studies revealed that PKCβ is required for cell-membrane translocation of CD25, but also transcriptional expression of CD25 via NF-κB activation. Therefore, PKCβ act as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors. In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. Figure Disclosures Zammarchi: ADC Therapeutics: Employment. Van Berkel:ADC Therapeutics: Research Funding. Melnick:Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy. Luger:Celgene: Research Funding; Cyslacel: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Agios: Honoraria; Ariad: Research Funding; Biosight: Research Funding; Kura: Research Funding; Onconova: Research Funding; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria. Meffre:AbbVie: Consultancy, Other: Grant. Weinstock:Celgene: Research Funding.

Abstract Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through β- and γ-, but not α-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds the B-cell receptor (BCR) to regulate its activity. Suggesting IL2-independent functions, defects in CD25-/-B-cells were not replicated in IL2-deficient mice. CD25 bound the BCR but not IL2Rβ- and IL2Rγ-chains. IL2Rβ- and IL2Rγ-chains can pair with other chains to form receptors for different cytokine-ligands. However, CD25 represents the first example of a cytokine receptor chain that binds to the BCR for negative feedback regulation. Likewise, in T-cells, CD25 had a bifunctional role and either functioned as IL2 receptor chain or as negative feedback regulator of T-cell receptor signaling. CD25-function was regulated by cell-membrane translocation, which required phosphorylation of its cytoplasmic tail at S268 (see schematic, left). In a family with monogenic autoimmunity, a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268 motif. CD25-interactome analyses identified PKCd as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors. In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. Figure. Figure. Disclosures Forman: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Weinstock:Genentech/Roche, Monsanto: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Travera: Equity Ownership; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties. Uzel:Novartis: Research Funding.

A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies. Here, we show that recombinant Ig (rIg) cloned from sort-purified single tumor cells from lipid-reactive sporadic and GD-associated gammopathy specifically bound lysolipids. Liposome sedimentation and binding assays confirmed specific interaction of lipid-reactive monoclonal Ig with lysolipids. The clonal nature of lysolipid-binding Ig was validated by protein sequencing. Gene expression profiling and cytogenetic analyses from 2 patient cohorts showed enrichment of nonhyperdiploid tumors in lipid-reactive patients. In vivo antigen-mediated stimulation led to an increase in clonal Ig and plasma cells (PCs) in GD gammopathy and also reactivated previously suppressed antigenically related nonclonal PCs. These data support a model wherein antigenic stimulation mediates an initial polyclonal phase, followed by evolution of monoclonal tumors enriched in nonhyperdiploid genomes, responsive to underlying antigen. Targeting underlying antigens may therefore prevent clinical MM.