IFITM3-Mediated Regulation of Cell Membrane Dynamics Is Essential for Malignant B-Cell Transformation

Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts. Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to "trap" intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, 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=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. 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. While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. 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 NRASG12D. 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 NRASG12D 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 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 (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization. Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. Figure Figure. Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.