IFITM3 (CD225) Regulates CD19 Surface Expression and CD19-Mediated Activation of PI3K Signaling in Pre-B Acute Lymphoblastic Leukemia Cells

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 on mouse and human B cells. However, besides its interaction with CD19, specific functions of Ifitm3 in normal B cells development and, potentially leukemogenesis, are not known. Recent approaches based on CD19-specific chimeric antigen receptors (CAR) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (e.g. Grupp et al., N Engl J Med 2013). However, in some cases, CD19 CAR treatment was followed by ALL relapse developing from a clone that lacked CD19 surface expression. Therefore, we studied factors that can potentially regulate CD19 surface on normal and leukemic pre-B 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 near-complete 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. Reconstitution of IFITM3 in patient-derived pre-B ALL cells rescued both CD19 expression and increased phosphorylation of CD19-Y⁵¹³ together with downstream SRC, SYK, PI3K signaling. Co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT, pointing to a central role of IFITM3 in regulating CD19/PI3K-AKT signaling in pre-B cells and pre-B ALL.

Deletion of Ifitm3 also resulted in significant cell cycle arrest in the G₀/G₁ phase (P<0.001) and increased protein levels of p53 and p21 compared to wild-type pre-B ALL cells. Moreover, treatment with low dose adriamycin (25 ng ml^-1) for induction of modest DNA damage significantly induced cellular senescence in Ifitm3^-/- but not wild-type pre-B ALL cells. Consistent with Ifitm3-mediated proliferative defects of Ph⁺ ALL cells, Ifitm3^-/- Ph⁺ ALL cells exhibited reduced self-renewal capacity (P=0.0004) decreased colony in colony forming assay compared to wild-type cells. Furthermore, Ifitm3 deficient B cell progenitors also showed significant inhibition of proliferation with inhibition of phosphorylation of Stat5^Y694and c-Myc expression.

4-hydroxytamoxifen (4-OHT)-inducible activation of CD19 rescued proliferative defects in Ifitm3 deficient B cell progenitors with release from G₀/G₁ cell cycle arrest associated with upregulation of phosphorylation of Stat5 ^Y694 and c-Myc expression. In addition, we found that CD19 positively regulates the surface expression of IL7R in B cell progenitors. Interestingly, CD19^low population in Ifitm3 deficient B cell progenitors showed lower surface expression of IL7R compared to CD19^high population. In contrast to B cell progenitors, forced expression of CD19 did not increase the proliferation of wild-type Ph⁺ ALL cells. Surprisingly, upregulation of CD19 induced apoptosis in Ifitm3-deficient Ph⁺ ALL cells with significant inhibition of BCR-ABL1 activity, phosphorylation of Stat5^Y694and Bcl2 expression.

Conclusion: These findings identify novel role of IFITM3 in regulating CD19 surface expression and CD19-Y⁵¹³ mediated PI3K-AKT signaling in pre-B cells and pre-B ALL. Preliminary experiment showed that agonistic antibodies against IFITM3/CD225 increased CD19/PI3K-AKT signaling and proliferation in pre-B ALL cells and future studies will elucidate whether blocking antibodies (e.g. decoy Fc-fusion molecules) will have useful effects in targeting CD19/PI3K-AKT signaling in human pre-B ALL.