▪Background and hypothesis: CD25 (IL2RA, interleukin 2 receptor α chain) is a transmembrane protein with a 13aa cytoplasmic tail. CD25 cooperates with β- and γ-chains in binding IL-2, but does not contribute to cytokine signaling. During normal B cell development, CD25 is specifically upregulated on the surface of IL7-dependent pre-B cells and is also expressed on the surface of a subset of human pre-B ALL cases. CD25-expressing ALL is typically associated with poor clinical outcome. For these reasons, we studied the functional significance of CD25 expression on human pre-B ALL cells.Results: Flow cytometry and immunohistochemistry staining on a large panel of patient samples (n=416; MDACC, ECOG) revealed specific cell surface expression of CD25 in Ph+ ALL and Ph-like ALL, which are both high-risk subtypes of ALL. In agreement with selective expression on high-risk subsets, high expression levels of CD25 at the time of diagnosis were predictive of poor overall clinical outcome in these studies (P=0.005). BCR-ABL1 in Ph+ ALL and related tyrosine kinases in Ph-like ALL strongly activate STAT5, which then induces transcriptional activation of the IL2RA locus. Since Stat5 is also active during normal pre-B cell differentiation, we first analyzed B cell development in Il2ra-/- mouse bone marrow. Il2ra-/- B cell development was blocked at the pre-B cell stage, consistent with specific upregulation of CD25 on pre-B cells. In human Ph+ ALL cells, we found IL2RB and IL2RG were not co-expressed with CD25, suggesting a function of CD25 in Ph+ALL that is distinct from IL2 signaling. To test the biological significance of tyrosine kinase/STAT5-induced activation of CD25, we developed an Il2ra-/- mouse model for BCR-ABL1 pre-B ALL. Interestingly, the cytoplasmic tail of CD25 includes phosphorylation sites (S268 and T271) that are known substrates for serine/threonine phosphorylation by PKCα, which was reported to regulate protein phosphatase 2A (PP2A). To investigate interacting proteins with the cytoplasmic tail of CD25, we performed immune precipitation (IP) against the flag-tagged CD25-tail in primary Ph+ ALL cells which were transduced with either a CD25-tail-flag or an EV-flag vector. 2D mass spectrometry and Western blot on the IP products confirmed strong interactions with PKCα and PP2A. Weestern blot analysis confirmed additional interactions with inhibitory phosphatases including PTEN, PTPN6 (SHP1) and Inpp5d (SHIP1) in human Ph+ALL cells. In addition, both 2D MS and Westernblot showed recruitment of the Stat5-feedback inhibitors CISH, SOCS2 and SOCS3 at the CD25 cytoplasmic tail.Studying functional parameters of Il2ra-/-BCR-ABL1 ALL cells, we found impaired proliferation and colony formation capacity and drastically increased increased phosphorylation levels of pABLY412, pSTAT5Y694, pERKT202/Y204, pAKTS473, pP38T180/Y182 and p53. Reconstitution of CD25 expression restored normal phosphorylation levels of these molecules, as well as proliferation and colony formation.In a serial transplant setting, we observed that leukemia initiation in transplant recipients from Il2ra-/-BCR-ABL1 ALL cells required 10- to 100-times higher cell numbers, suggesting that CD25 contributes to leukemia initiation. In addition, CD25 expression is associated with a higher level of drug-resistance: In patient-derived pre-B ALL cells with mixed CD25Low and CD25High populations, the standard chemotherapy agent vincristine selectively induced apoptosis of in CD25Low but not CD25High ALL cells. An anti-CD25 immunotoxin drugs efficiently eradiated CD25High leukemia cells and thereby overcame drug-resistance against vincristine.Conclusions: Our studies identified CD25 as a surface receptor that mediates membrane recruitment of PP2A and CISH, SOCS2, negative feedback regulators of STAT5. CD25 is transcriptionally activated by STAT5 and therefore specifically expressed on high-risk ALL subtypes with oncogenic activation of the Stat5 pathway (Ph+ ALL and Ph-like ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.