PKCδ-Mediated Phosphorylation of CD25 Initiates Feedback Control of Oncogenic Tyrosine Kinases in Acute Lymphoblastic Leukemia

Background and Significance: CD25 (IL2RA) has been extensively studied as one of three chains of the IL2-receptor expressed on T- and NK-cells. However, CD25 is also highly expressed in a subset of B-ALL cases, the significance of which was unknown.

Results: Studying CD25 mRNA and surface expression in three clinical cohorts for children (COG P9906, n=207; AALL0232, n=1,104) and adults (ECOG E2993, n=165), we found that CD25 mRNA and surface expression (flow cytometry) were consistently associated with decreased overall (P=0.016) and relapse-free (P=2.2E-04) survival. Instead of IL2-signaling, high expression levels of CD25 were significantly enriched among B-ALL cases carrying oncogenic tyrosine kinases including BCR-ABL1 in Ph+ B-ALL and mutations of FLT3, JAK1, JAK2, JAK3, NTRK3 in Ph-like B-ALL (χ² test P=2.2 E-09). Studying inducible models for oncogenic BCR-ABL1 and FLT3 tyrosine kinases in murine B-cell progenitors, we found that CD25 surface levels were upregulated by 15- to 40-fold within six hours of doxycycline-mediated induction.

Modeling CD25-deletion in the context of oncogenic tyrosine kinase signaling in murine CD25-fl/fl B-ALL cells revealed that Cre-mediated ablation of CD25 expression resulted in cell cycle arrest, loss of colony formation capacity and acute cell death. Upon Cre-mediated deletion of CD25, BCR-ABL1 B-ALL lost their ability to initiate leukemia in transplant recipient mice. Likewise, genetic deletion of CD25 in patient-derived xenografts from Ph+ B-ALL resulted in loss of colony formation capacity and cell death. Cre-mediated excision of CD25 exons 2-3 resulted in expression of a truncated non-functional CD25 transcript, which was 12-fold upregulated along with negative feedback regulators of NF-κB, Nfkbia, Nfkbie, Nfkbiz (6- to 21-fold). Mass spectrometry-based global phosphoproteomic analyses showed that Cre-mediated deletion of CD25 resulted in massive increases of SYK, FYN, JAK3 and ERK phosphorylation as well as phosphorylation of PKCδ and NF-κB components. Conversely, CD25-deletion caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Imbalances of signal transduction upon CD25-deletion were confirmed by Western blot, namely hyperphosphorylation of SYK (Y348/Y352), ERK (T202/Y204), PKCδ (T505) and NFKBIA (S32/S36) as well as loss of phosphorylation of the SHP1 (Y564) and SHIP1 (Y1020) phosphatases. In addition to Cre-mediated deletion of CD25 in murine B-ALL cells, we confirmed these results by CRISPR-mediated deletion of CD25 in patient-derived xenograft (PDX) Ph+ B-ALL cells.

Mechanistic studies revealed that oncogenic tyrosine kinase signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail (S268, T271). Genetic deletion of PKCδ and mutations of S268/T271 residues demonstrated that PKCδ-mediated CD25-phosphorylation was critical for CD25 surface expression and CD25-dependent feedback control of oncogenic tyrosine kinase signaling. PKCδ activation downstream of oncogenic tyrosine kinases initiates negative feedback regulation via phosphorylation and cell surface expression of CD25. This step was required for recruitment and activation of SHP1 and SHIP1-phosphatases to curb oncogenic signaling strength. Interactome analyses based on proximity labeling and mass-spectrometry, co-immunoprecipitation and molecular dynamics simulations revealed that the phosphorylation of CD25 cytoplasmic tail is required and sufficient for its interaction with PKCδ, as well as recruitment and activation of SHP1 and SHIP1. Since tyrosine kinase-driven B-ALL cells critically depend on CD25-mediated feedback control, we tested a recently developed CD25 antibody-drug conjugate (ADC) in a preclinical model based on refractory Ph+ B-ALL PDX. While mice receiving control ADC died after 60 days, four injections with CD25-ADC induced complete remission in NSG mice. These mice survived indefinitely, suggesting that CD25-ADC may be effective to induce lasting remissions in patients with refractory Ph+ B-ALL.

Conclusions: Our findings highlight the unexpected function of CD25 as a previously unrecognized feedback regulator of oncogenic tyrosine kinase signaling. Since CD25-mediated feedback control is essential for tyrosine kinase-driven B-ALL, targeting CD25 may be a promising strategy in the treatment of Ph+ and Ph-like B-ALL.