IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies
2016
Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection.
Endosomal membranelocalized IFITM3 appear to prevent fusion events of intraluminal viral particles to the
endosomal membranethrough accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell
co-receptorsincluding
CD19,
CD81and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen
CD19. Although 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), in some cases,
CD19-specific engineered
chimeric antigen receptors(CART19) treatment was followed by ALL relapse developing from clones that lacked
CD19surface expression. Results: Studying IFITM3 mRNA levels in B cell lineage 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, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and 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 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of
CD19expression 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 signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice. In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with
CD19,
LYN,
SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of
CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers
CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through
Filipinbased cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of
lipid raftsformation with decreased levels of ganglioside GM1. Thereby, Inadequate
CD19/BCR
co-receptorsignaling caused by disruption of
lipid raftshomeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo . Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining
lipid raftsand
CD19surface expression. IFITM3-dependent
lipid raftstability and
CD19surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of
CD19surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. Disclosures No relevant conflicts of interest to declare.
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