Ifitm3 Is Essential for PI(3,4,5)P3-Dependent B-Cell Activation and Leukemogenesis

Background: The interferon-inducible transmembrane protein Ifitm3 (also known as Fragilis) plays an important role in primordial germ cell specification and functions as critical antiviral effector preventing fusion of virion-containing vesicles with endosomal membranes.

Results: Here, we identified Ifitm3 as a biomarker of poor clinical outcome in patients with various B-cell malignancies. We found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.011; n=207) and adults (ECOG E2993; P=0.017; n=55) with B-ALL. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B-ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion.

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^G12D. Strikingly, deletion of Ifitm3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3^-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRAS^G12D and failed to initiate fatal leukemia in transplant recipient mice. 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 mechanistic studies, we identified N-terminal phosphorylation at endocytic motif (²⁰YEML²³) by Src-kinases induced Ifitm3 cell surface accumulation during normal activation or oncogenic transformation of B-cells. In a lipid-binding assay in vitro, recombinant IFITM3 directly bound to PI(3,4,5)P₃ but not any other phospholipids. In the cell membrane, therefore, IFITM3 functioned as a scaffold for PI(3,4,5)P₃ and was essential for Src-kinase and PI3K signaling, as well as lipid raft formation and surface expression of multiple raft-associated receptors. Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of Src-kinase signaling. In the absence of Ifitm3, resting B-cell populations developed normally. However, consistent with defective Src-kinase and PI3K signaling, Ifitm3^-/- B-cells failed to form germinal centers and to give rise to antigen-specific humoral immune responses. Likewise, Ifitm3^-/- B-cell precursors were resistant to malignant transformation and lacked the ability to initiate BCR-ABL1- and NRAS^G12D-driven leukemia. Conversely, an Ifitm3-phosphomimetic of Src-kinase phosphorylation induced constitutive cell membrane localization, triggered oncogenic Src-PI3K signaling and initiated overt leukemia in pre-malignant B-cells.

Conclusions: We conclude that Src-kinase-mediated phosphorylation of Ifitm3 induces a dynamic switch from antiviral effector functions in endosomes to lipid raft signaling at the cell membrane. While membrane-bound Ifitm3 is critical for normal B-cell receptor signaling and antigen-specific B-cell responses, its role as signal amplifier can be leveraged by multiple oncogenes for malignant B-cell transformation.

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