Ma, Ning

CD25 is a subunit of the interleukin-2 (IL-2) receptor on T cells and natural killer (NK) cells. Acute leukemias with oncogenic tyrosine kinases often include CD25 + leukemia subpopulations, which portend poor clinical outcomes for patients; however, acute leukemia cells do not respond to IL-2. Here, we identified CD25 and its phosphorylation by protein kinase Cδ (PKCδ) as central elements of a feedback loop that stabilized fluctuations in oncogenic tyrosine kinase signaling in acute lymphoblastic and myeloid leukemia. Genetic ablation of CD25 in murine and patient-derived xenograft (PDX) models of acute leukemias reduced clonal fitness, colony formation, and leukemia-initiation capacity in serial transplant recipients. Oncogenic tyrosine kinase signaling in leukemia cells stimulated NF-κB–mediated CD25 expression, whereas PKCδ-mediated phosphorylation of CD25 suppressed oncogenic tyrosine kinase signaling through inhibitory phosphatases, such as PTPN6. Interactome analyses and mass spectrometry–based global phosphoproteomic analyses showed that CD25 deletion abolished the phosphatase activity of PTPN6, resulting in enhanced activation of tyrosine kinases and NF-κB. Four injections of a CD25 antibody-drug conjugate induced complete remission in mice transplanted with PDX refractory leukemia. These findings highlight the dependency of tyrosine kinase–driven leukemias on robust feedback control and the role of PKCδ and CD25 in assembling its components.

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.