Identification of a Conserved Intracellular Loop (CIL) Structure That Scaffolds PIP3 to Amplify Oncogenic Signaling during Malignant B-Cell Transformation

ABSTRACT

Background: Within seconds of antigen-encounter, B-cell receptor (BCR) signaling induces dramatic changes of cell membrane lipid composition, including >40-fold increases of local PIP3-concentrations within lipid rafts. While several structural elements, including pleckstrin homology (PH) domains have been identified as PIP3-binding proteins, the underlying mechanisms that amplify BCR-signaling to assemble large signaling complexes within lipid rafts within 15 to 30 seconds, remained elusive. To understand the mechanistic and biophysical requirements for PIP3 accumulation during normal B-cell activation and acute oncogenic transformation, we identified PIP3-interacting proteins by cell-surface proteomic analyses. Results: In addition to proteins known to bind PIP3 with their PH-domains, we identified the short 133 aa protein IFITM3 (interferon-inducible transmembrane protein 3) as a top-ranking PIP3 scaffold. This was unexpected because IFITM3 was previously identified as endosomal protein that blocks viral infection by stiffening endosomal membranes to firmly contain viral cargo. Previous studies revealed that polymorphisms that lead to the expression of truncated IFITM3 are associated with increased susceptibility to viral infections, including SARS-CoV2. Among known cell membrane lipids, PIP3 has the highest negative charge. Instead of a PH-domain, IFITM3 laterally sequestered PIP3 through electrostatic interactions with two basic lysine residues (K83 and K104) located at the membrane-solution interface. Together with three other basic lysine and arginine residues K83 and K104 form a conserved intracellular loop (CIL), which enable IFITM3 to efficiently capture two PIP3 molecules. Bivalent PIP3-binding of the IFITM3-CIL enables a crosslinking mechanism that results in dramatic amplification of B-cell activation signals and clustering of large signaling complexes within lipid rafts. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell activation and oncogenic kinases induced phosphorylation at IFITM3-Y20, resulting in translocation of IFITM3 from endosomes and massive accumulation at the cell surface. Ifitm3ˉ /ˉ naïve B-cells developed at normal numbers, however, activation by antigen encounter was compromised. In Ifitm3ˉ /ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors and impaired PI3K-signaling. Ifitm3ˉ /ˉ B-cells were unable to undergo affinity maturation and di not contribute to germinal center formation upon immunization. Analyses of gene expression and clinical outcome data from patients in six clinical cohorts for pediatric and adult B-ALL, mantle cell lymphoma, CLL and DLBCL, we consistently identified IFITM3 as a top-ranking predictor of poor clinical outcome. Inducible activation of BCR-ABL1 and NRAS G12D rapidly induced development of B-ALL but failed to transform and initiate B-ALL from Ifitm3ˉ /ˉ B-cell precursors. Conversely, the phospho-mimetic IFITM3-Y20E mutation, mimicking phosphorylation of the IFITM3 N-terminus at Y20 induced constitutive membrane localization of IFITM3, spontaneous aggregation of large oncogenic signaling complexes and readily initiated transformation in a genetic model of pre-malignant B-cells. Conclusions: We conclude that phosphorylation of IFITM3 upon B-cell activation induces a dynamic switch from antiviral effector functions in endosomes to oncogenic signal-amplification at the cell-surface. IFITM3-dependent amplification of PI3K-signaling is critical to enable rapid expansion of activated B-cells. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation and initiation of B-lymphoid leukemia and lymphoma. [Formula presented] Disclosures Weinstock SecuraBio Consultancy;ASELL Consultancy;Bantam Consultancy;Abcuro Research Funding;Verastem Research Funding;Daiichi Sankyo Consultancy, Research Funding;AstraZeneca Consultanc ;Travera Other Founder/Equity;Ajax Other Founder/Equity.