Background and hypothesis: CD25 (IL2RA) represents the α chain of the interleukin 2 receptor on T cells and plays an important role in the maintenance of regulatory T (Treg) cells, hence preventing T cell autoimmunity. In a comprehensive gene expression analysis, we found that CD25 is specifically upregulated by pre-B cell receptor (pre-BCR) signaling during early B cell development and oncogenic tyrosine kinase that mimic pre-BCR signaling (e.g. in Ph+ ALL and Ph-like ALL). In adults with Ph+ ALL (ECOG; MDACC) and children with Ph-like ALL (P9906) patients with CD25 expression at the time of diagnosis have a particularly poor outcome (n=416; P=0.005). For these reasons, we studied the function of CD25 in B cell development and leukemia in a series of genetic experiments.
Results: Unlike T cells, CD25 (IL2RA) does not function as IL2 receptor chain in B cells and B-lineage ALL: CD25 expressed on B-lineage cells did not pair with IL2Rb and g-chains and was not responsive to IL2. Il2ra-/- B cells were arrested at the pre-B cell stage with hyperactive pre-BCR downstream signaling including SRC, BTK and ERK. In the presence of CD25, Il2ra+/+ B cells responded to engagement of the pre-BCR with phosphorylation of pre-BCR downstream tyrosine kinases and coordinated release of Ca2+ from cytoplasmic stores. In the absence of CD25 (Il2ra-/-), the pre-BCR signals autonomously, resulting in uncoordinated Ca2+ oscillations of variable duration. While CD25 does not function as IL2 receptor chain in B cells, it coordinates pre-BCR-dependent signal transduction and regulates its intensity.
The pre-BCR related tyrosine kinase BTK is phosphorylated by BCR-ABL1 in Ph+ ALL and other tyrosine kinase oncogenes in Ph-like ALL (Chen et al., 2015). Interestingly, overexpression of a constitutively active form of BTK resulted in strong upregulation of CD25 surface expression. Conversely, the BTK-inhibitor ibrutinib abolished CD25 expression suggesting that feedback control between pre-BCR signaling and CD25 requires BTK.
The ability of CD25 to stabilize oncogenic signaling strength in Ph+ ALL and Ph-like ALL was important for leukemia-initiation and development of fatal disease. In the absence of CD25, Il2ra-/- ALL cells showed impaired proliferation and colony formation. Serial transplantation experiments revealed a profound defect of Il2ra-/- ALL cells to initiate leukemia. 100-times more cells were required to cause fatal disease. In addition, CD25 expression mediated drug-resistance in ALL cells: In patient-derived pre-B ALL cells with heterogeneous CD25 expression, vincristine selectively induced apoptosis in CD25Low cells but spared CD25High ALL cells. Combination with an anti-CD25 immunotoxin efficiently eradiated CD25High leukemia cells and sensitized the ALL cell population to treatment with vincristine.
To elucidate the mechanism of how CD25 coordinates negative feedback control of pre-BCR signaling or its oncogenic mimics, we focused on its short (13aa) cytoplasmic tail, which includes two phosphorylation sites (S268 and T271) that are known substrates for serine/threonine protein kinase, PKCα. To identify cytoplasmic interaction partners of CD25, we overexpressed a Flag-tagged truncated form of CD25 including a myristoylation signal for constitutive membrane localization, transmembrane domain and cytoplasmic tail. Immunoprecipitation (IP; Flag) followed by 2D mass spectrometry revealed strong interactions of PP2A with cytoplasmic tail of CD25. Western blots showed additional strong interactions of the cytoplasmic tail of CD25 with inhibitory phosphatases PTEN, SHP1 and SHIP1. Importantly, reconstitution of myristoylated CD25 tail but not a mutant construct lacking the serine/threonine motif (S268A/T271A) rescued proliferation and survival defects of Il2ra-/- ALL cells.
Conclusion: We identified CD25 as a surface receptor that mediates membrane recruitment of PP2A, PTEN, SHP1 and SHIP1, which balances fluctuations in signaling output from a pre-B cell receptor or its oncogenic mimic in ALL cells (e.g. BCR-ABL1 in Ph+ 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.