Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.