Article IFITM3 functions as a PIP3 scaffold to amplify PI3K signalling in B cells

­­­­­ https://doi.org/10.1038/s41586-020-2884-6 Jaewoong Lee1, Mark E. Robinson1, Ning Ma2, Dewan Artadji1, Mohamed A. Ahmed3, Gang Xiao3, Teresa Sadras1, Gauri Deb3, Janet Winchester3, Kadriye Nehir Cosgun1, Received: 11 September 2019 Huimin Geng4, Lai N. Chan1, Kohei Kume1, Teemu P. Miettinen5,6, Ye Zhang5, Matthew A. Nix4, Accepted: 13 August 2020 Lars Klemm1, Chun Wei Chen3, Jianjun Chen3, Vishal Khairnar3, Arun P. Wiita4, Andrei Thomas-Tikhonenko7, Michael Farzan8, Jae U. Jung9, David M. Weinstock10,11, Published online: 4 November 2020 Scott R. Manalis5,12, Michael S. Diamond13,14,15, Nagarajan Vaidehi2 & Markus Müschen1,16 ✉ Check for updates

Interferon-induced transmembrane protein 3 (IFITM3) has previously been identifed as an endosomal protein that blocks viral infection1–3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identifed IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Iftm3−/− naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specifc antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Iftm3−/− B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scafold and central amplifer of PI3K signalling. The amplifcation of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scafold for the accumulation of PIP3. In Iftm3−/− B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral efector functions in endosomes to a PI3K amplifcation loop at the cell surface. IFITM3-dependent amplifcation of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high afnity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.

IFITM3 is a small antiviral membrane protein (133 amino acids in size) encode a 21-amino-acid truncation of the N terminus (which includes with activity against influenza A virus, severe acute respiratory syn- Y20), although a recent transcript analysis found no evidence for such drome (SARS) coronavirus and many other viruses1–5. Endocytosis processed mRNA9. IFITM3 probably functions as an antiviral effector and preferential endosomal localization of IFITM3 are mediated by molecule by limiting viral fusion through increasing the ‘rigidity’ of its N-terminal AP2 sorting motif, YEML1. Phosphorylation of this motif endosomal cell membranes10,11. IFITM3 is overexpressed in multiple at Y20 by SRC kinases prevents not only endocytosis but also protea- types of cancer, and its expression levels correlated with histopatho- somal degradation6,7. Individuals with the IFITM3 single-nucleotide logical grading and staging12,13. Although IFITM3-dependent antiviral polymorphism (SNP) rs12252-C allele show increased morbidity and effector functions are restricted to endosomal compartments14, we mortality from H1N1 strains of influenza A virus8 and SARS coronavi- report here on IFITM3-dependent functions at the cell surface of rus 24. IFITM3 SNP rs12252-C was predicted through mRNA splicing to B cells, where IFITM3 functions as amplifier of PI3K signalling.

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Nature | Vol 588 | 17 December 2020 | 491 Article between CD19 and LYN17, as CD19 was not phosphorylated and did not IFITM3 is essential for B cell affinity maturation interact with LYN. IFITM3-deficient MCL cells expressed lower levels of Although IFITM3 is only minimally expressed in normal resting B cells, LYN and did not activate SRC kinases in response to BCR engagement BCR engagement and acute activation of the PI3K pathway strongly (Extended Data Fig. 2e). RNA-sequencing gene-expression studies induced the expression of this protein (Extended Data Fig. 1a–c). Expres- revealed upregulation of Ifitm1 in Ifitm3−/− B-ALL cells, presumably to sion of IFITM proteins has recently been established as a diagnostic compensate for loss of Ifitm3. Gene set enrichment analyses showed biomarker of high-risk B cell-lineage acute lymphoblastic leukaemia that Ifitm3 deficiency was associated with reduced expression of com- (B-ALL) that expresses the oncogenic BCR–ABL1 tyrosine kinase or ponents of the BCR signalling pathway and a phenotype reminiscent related kinases15; we found that BCR–ABL1 kinase inhibition decreased of B cell anergy (Extended Data Fig. 3a–c). levels of IFITM3 expression (Extended Data Fig. 1d). Levels of IFITM3 mRNA were generally higher in B cell malignancies than in their normal counterparts (Extended Data Fig. 1f, g) and elevated IFITM3 mRNA Effect of phosphorylation of IFITM3 at Y20 levels were associated with poor clinical outcomes in multiple clini- Combinations of surface and intracellular staining revealed the cyto- cal cohorts of patients with B cell malignancies (including paediatric plasmic localization of the N terminus of IFITM3, with a short extra- and adult B-ALL and mantle cell lymphoma) (Extended Data Fig. 1h–l). cellular portion of the C terminus (Extended Data Fig. 3d). Previous IFITM3 was previously identified as a transcriptional target of the tran- studies have shown that phosphorylation of IFITM3 at Y20 prevents scriptional repressor IKZF116, which suggests that high levels of IFITM3 both endocytosis and degradation6,7. Phosphomimetic IFITM3(Y20E) expression may be the result of genetic lesions in IKZF1. Our results was constitutively localized at the cell surface. Similarly, phospho- confirmed that reconstitution of IKZF1 suppresses IFITM3 expression, rylation by BCR–ABL1 induced the accumulation of IFITM3 at the cell and that inducible deletion of Ikzf1 in mice relieves transcriptional membrane, in a process that was sensitive to inhibition of BCR–ABL1 repression (Extended Data Fig. 1m, n). However, a multivariate analysis or SRC kinases (Extended Data Fig. 3e). Therefore, the phospho- of risk factors in B-ALL showed that high IFITM3 mRNA levels are an rylation of Y20 induces a dynamic switch from antiviral functions in independent predictor of poor outcome, regardless of IKZF1 deletion endosomes to BCR or oncogene signalling at the cell surface (Extended (Extended Data Fig. 1o). We next examined how the genetic deletion Data Fig. 3e, f). To test whether phosphorylation of IFITM3 at Y20 and of Ifitm3 affects normal B cell development in mice. Surface expres- constitutive membrane localization are sufficient to enable onco- sion of CD19—a common B cell surface receptor that promotes SRC genic signalling, we overexpressed wild-type and IFITM3(Y20E) in kinase and PI3K signalling17—was substantially diminished in Ifitm3−/− premalignant B cells carrying a BCR-ABL1-knock-in allele, which did not pre-B cells (Fig. 1a). Consistent with increased apoptosis and reduced readily give rise to leukaemia20. Expression of IFITM3(Y20E) increased proliferation, expression of MYC and BCL2 as well as PI3K signalling the ability of pre-B cells to form colonies by 129-fold (P = 2.1 × 10−8) were reduced in Ifitm3−/− pre-B cells (Fig. 1a–c). The development of (Fig. 2g), induced oncogenic signalling and initiated fatal leukaemia resting Ifitm3−/− B cell populations was largely unperturbed (Extended in congenic transplant recipient mice (Fig. 2h–j). To elucidate how Data Fig. 2), whereas B1 cell and marginal-zone B cell populations were membrane-bound IFITM3(Y20E) promotes malignant transformation, decreased (Fig. 1d, Extended Data Fig. 2). To assess B-cell-specific effects we performed phosphoproteomic analyses of IFITM3 Y20 signalling in of IFITM3 during humoral immune responses, we performed adoptive patient-derived B-ALL cells. Consistent with processive signal amplifi- transfer experiments: Ifitm3+/+ or Ifitm3−/− splenic B cells were trans- cation17, engagement of IFITM3 induced phosphorylation of CD19 on ferred into congenic μMT mice that lack endogenous mature B cells. multiple residues—including Y531, which mediates PI3K activation. In After reconstitution of mature B cell development, recipient μMT mice addition, Y20 of IFITM3 engaged multiple components of the BCR and were immunized with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP) integrin receptor signalling pathways (Fig. 3a, Extended Data Fig. 4a), conjugated to keyhole limpet haemocyanin (KLH) (NP–KLH). Upon which converge with CD19 at the level of PI3K activation. A compre- immunization, μMT mice reconstituted with Ifitm3+/+ B cells developed hensive analysis of phosphosites showed a significant enrichment of about 5% NP-specific germinal-centre B cells, which were largely absent phosphorylation in the PI3K, BCR and integrin receptor pathways, in μMT mice reconstituted with Ifitm3−/− B cells (Fig. 1e–h). Similarly, which we confirmed for SRC, CD19 and PI3K signalling by western blot the number of follicles in the spleen, PNA+ germinal-centre B cells (Fig. 3a–c). IFITM3(Y20E) required crosslinking by a full antibody, as and production of IgM, IgG1 and IgG2b antibodies were reduced in the engagement of Y20 of monomeric IFITM3 using F(ab) fragments the absence of Ifitm3 (Fig. 1i). These abnormalities in Ifitm3-deficient did not elicit PI3K activation (Extended Data Fig. 4b). Because PI3K B cells phenocopy B cell defects seen in mice upon deletion of Pik3cd18 signalling in mouse B-ALL cells increased Ifitm3 mRNA levels by 10-fold and Cd1919. and protein levels by more than 50-fold, these observations suggest a feed-forward loop of signal amplification between IFITM3 and PI3K signalling (Extended Data Fig. 1b–e). Comparable to effects on BCR Role of Ifitm3 in oncogenic signalling signalling and CD19 surface expression in B cells, genetic deletion of To elucidate a potential contribution of Ifitm3 to oncogenic signalling, IFITM3 in human Jurkat T cells compromised T cell receptor signalling we studied the transformation of Ifitm3+/+ and Ifitm3−/− pre-B cells by and CD3 surface expression (Extended Data Fig. 4c), which suggests BCR-ABL1 and NRASG12D (Fig. 2a, b). As in normal B cells, Ifitm3−/− leukae- that IFITM3 may have a similar role in T cells. mia cells in a mouse model of B-ALL (Ifitm3−/− B-ALL cells) showed multi- ple defects in survival and proliferation, and a markedly reduced ability to form colonies in semi-solid methylcellulose (Fig. 2b, c). Consistent IFITM3 enables CD19–PI3K signalling with a 70-fold reduction of the frequency of leukaemia-initiating cells By enriching for cell-surface proteins using N-linked glycosylation-site (P = 3.7 × 10−7), Ifitm3−/− B-ALL cells did not initiate overt leukaemia in biotin labelling, we identified 65 surface receptors—most of which were immunodeficient transplant recipient mice (Fig. 2d, e). Similar to B-ALL associated with lipid rafts—that were downregulated in Ifitm3−/− B-ALL cells, deletion of IFITM3 in mantle cell lymphoma (MCL) cells reduced cells, including CD19, BCR-associated and adhesion receptors. Only competitive fitness and proliferation (Fig. 2f). Both mouse Ifitm3−/− five surface proteins were upregulated—including the IFITM family B cells and human IFITM3-deficient MCL cells showed defective Ca2+ member IFITM2, which suggests a possible compensation mechanism release in response to BCR engagement, and partially lost CD19 surface (Extended Data Fig. 4d). We performed validation by flow cytometry expression (Extended Data Fig. 2c, d). In addition, BCR engagement for 17 surface receptors for IFITM3 loss-of-function (Ifitm3−/−) and in IFITM3−/− MCL cells did not induce processive signal amplification gain-of-function (IFITM3(Y20E)) (Extended Data Fig. 4e–g). Continuous

492 | Nature | Vol 588 | 17 December 2020 abIfitm3+/+ Ifitm3–/– cdIfitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– MZB AKT-pS473 P = 1.2 × 10–4 CD23 CD19 AKT 609 174 8 Ifitm3+/+ 10 7 FSC CD21 per ml) 6 MYC 6 56 25 32 11 Total B1 4 p53 BrdU 2 p21 MAC1 Ifitm3–/– 17 52 0 21 6 B1a BCL2 Cell count ( × 10 0 12345 CD5 Annexin V Time (d) β-Actin

7-AAD IgM

e B220 CD3 PNA Overlay f Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/–

No transplant P = 9.93 × 10 6 P = 6.42 × 10 μMT 8 Non-immunized 6 4 cells (%) cells (%) –6 –5 + +/+ + Ifitm3 B cells 4 NP + μMT GL7 2 + Non-immunized 2 CD95 CD95 0 0 –/– e e e e Ifitm3 B cells NP NP NP NP μMT Vehicl Vehicl Vehicl Vehicl Non-immunized +/+ –/– +/+ –/– g Ifitm3 Ifitm3 Ifitm3 Ifitm3 P = 2.15 × 10 P = 1.6 × 10 )

) 1.0 4 Ifitm3+/+ B cells 1.0 4 μMT 0.8 NP–KLH

0.6 –5 –7 cells (×10 cells (×10 + 0.5 + 0.4 NP

–/– GL 7 Ifitm3 B cells + + μMT 0.2 NP–KLH CD95 CD95 0 0 e e e e NP NP NP NP Vehicl Vehicl Vehicl Vehicl h i IgM IgG1 IgG2b Non-immunized (vehicle) Gated on CD19+B220+ Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– +/+ –/– +/+ –/– 4 Ifitm3 Ifitm3 Ifitm3 Ifitm3 P = 1.5 × 10 P = 0.00049 P = 2.42 × 10 1.5 3 3 CD95 CD95

0.3 0.2 0.1 0.2 –5 1.0 2 –5 GL7 NP 2 450 nm Immunized (NP–KLH) Gated on CD19+B220+ OD Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– 0.5 1 1 CD95 CD95 0 0 0 6.4 0.2 5.5 0.2 e e e e e e NP NP NP NP NP NP GL7 NP Vehicl Vehicl Vehicl Vehicl Vehicl Vehicl

Fig. 1 | Ifitm3 is essential for B cell activation and affinity maturation in day 12 after immunization and subjected to immunofluorescence staining of germinal centres. a, CD19 surface expression (top), cell cycle progression tissue sections with B220, CD3 and peanut agglutinin (PNA). Scale bar, 500 μm. (middle) (percentages in S phase) and cell viability (bottom) (percentages of f–h, Splenocytes collected from μMT mice (n = 10) were analysed by flow annexin V+7-AAD+ cells) were measured. b, Number of viable Ifitm3+/+ and cytometry 12 days after immunization for CD95, GL7 and NP to identify Ifitm3−/− pre-B cells were counted at the times indicated. c, Levels of AKT NP-specific germinal-centre B cells. Relative fractions (f), absolute numbers phosphorylated at S473 (AKT-pS473), AKT, MYC, p53, p21 and BCL2 were (g) and representative flow cytometry plots (h) are shown. i, Levels of serum assessed in Ifitm3+/+ and Ifitm3−/− pre-B cells. d, Splenic B cells from Ifitm3+/+ and immunoglobulin isotypes in μMT recipient mice transplanted with Ifitm3+/+ or Ifitm3−/− mice (n = 5) were analysed for CD21highCD23low or CD21highCD23− Ifitm3−/− B cells are shown before and after immunization (n = 10; day 12). marginal-zone B cells (MZB), peritoneal-cavity B cells for MAC1+IgM+ B1 cells Serum levels of IgM, IgG1 and IgG2b were determined by enzyme-linked and CD5+IgM+ B1a cells. e, Splenic B cells from Ifitm3+/+ and Ifitm3−/− mice immunosorbent assay (ELISA). For gel source data for a–c, see Supplementary were adoptively transferred to μMT recipient mice (n = 10) followed by Fig. 1. In b, f, g, i, mean ± s.d. indicated; significance determined by two-tailed immunization with 0.5 mg of NP–KLH or vehicle. Spleens were collected on t-test. forced expression of CD19 (for more than one week) in Ifitm3−/− B cells were biotinylated on the basis of their proximity to the cytoplasmic partially restored defective survival and proliferation, which required BirA moiety, collected using streptavidin-coated beads and iden- the PI3K-activation motif Y531 of CD19 (ref. 21) (Extended Data Fig. 5a–e). tified by mass spectrometry. Consistent with phosphoproteomic However, inducible translocation of CD19 to the cell membrane did analyses (Fig. 3a), and across different cell types (B-ALL and MCL) not restore any defects in Ifitm3−/− B cells, which suggests that IFITM3 and stimulations (anti-HA and anti-IgM), IFITM3-interacting proteins is still required to integrate CD19 into signalling complexes for SRC included BCR, integrin receptor and PI3K signalling elements (Fig. 3d, kinase and PI3K signalling (Extended Data Fig. 5f–h). To study com- e, Extended Data Fig. 6a–d). We transduced MCL cells with Flag–IFITM3 plexes that interact with IFITM3, we performed enzyme-catalysed for validation by co-immunoprecipitation and western blotting (Fig. 3f, proximity labelling studies on the basis of N-terminal fusions of the g). Using proximity ligation assays, we confirmed the inducible for- BirA biotin ligase with IFITM3(Y20E) carrying an extracellular hae- mation of a complex between IFITM3 and the BCR signalling chain magglutinin (HA) tag in B-ALL and IgM+ MCL cells. After engagement CD79B upon engagement of the BCR. Interactions between BCR and with IFITM3 (anti-HA) or BCR (anti-IgM), IFITM3-interacting proteins IFITM3 were induced within minutes and dissociated after 30 min

Nature | Vol 588 | 17 December 2020 | 493 Article

a BCR-ABL1 B-ALL NRAS G12D B-ALL b BCR-ABL1 B-ALL NRASG12D B-ALL Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/–

AKT-pS473 CD19 1,049 537 11,757 5,151 AKT FSC 66 49 56 36 MYC p53 BrdU p21 16 49 14 48 BCL2

Annexin V β-Actin

7-AAD c BCR-ABL1 B-ALL NRASG12D B-ALL g LSL-BcrBCR-ABL1 × Mb1-cre Ifitm3+/+ Ifitm3–/– Ifitm3+/+ Ifitm3–/– EV IFITM3–HA Y20E–HA

267 86 250 12 5 470 644

P = 0.0004 P = 1.69 × 10–5 P = 1.0 × 10–5 P = 1.6 × 10–5

d 100 100 hj –/– LSL-BcrBCR-ABL1 × Mb1-cre Ifitm3 EV IFITM3Y20E 80 104 cells –/– 100 3 80 Ifitm3 P = 0.0001 EV CD19- 10 cells 105 cells 80 pY531 60 60 60 CD19 40 +/+ 40 IFITM3 Ifitm3 40 103 cells Survival (%) 20 4 20 SRC- 10 cells 20 pY416 Ifitm3+/+ Ifitm3–/– 105 cells Ifitm3+/+ 105 cells Y20E 0 0 0 0 30 60 90 120 0 40 80 120 100 150 200 250 LYN +/+ Ifitm3 Days after injection Days after injection Days after injection AKT- 105 cells pS473 AKT efIfitm3+/+ 1 in 621 LIC Mantle cell lymphoma i β-Actin Ifitm3–/– 1 in 43,259 LIC 1.0 LSL-BcrBCR-ABL1 × Mb1-cre 0 P = 3.7 × 10–7 gNT EV IFITM3–HA Y20E–HA Day –0.5 Ifitm3–/–

cells (%) 0.8 3 –1.0 + 68 –1.5 NT No. 1 No. 2

gRN A 0.6 + IFITM3 gIFITM3 no. 2 (log(fraction)) –2.0 +/+ 145 Ifitm3 β-Actin gIFITM3 no. 1 Non-leukaemic cells Cas9 0.4 176 0246810 0481216 Cell number (×105) Incubation time (d)

Fig. 2 | Essential role of IFITM3 in oncogenic signalling and B cell doxycycline (mean ± s.d.), and IFITM3 levels were measured by western blot. transformation. a, CD19 surface expression (top), cell cycle progression g, Premalignant LSL-BcrBCR-ABL1 × Mb1-cre pre-B cells expressing IFITM3, (middle) (percentages in S phase) and cell viability (bottom) (percentages of IFITM3(Y20E) or empty vector (EV) were plated for colony-forming assays annexin V+7-AAD+ cells) were measured. b, c, Ifitm3+/+ and Ifitm3−/− B-ALL cells (7 days). Representative images shown at 1× (top) and 10× (bottom) transformed with human BCR-ABL1 or NRASG12D were assayed for levels of magnification. h, Survival analyses (P = 0.0001, log-rank test) of congenic AKT-pS473, AKT, MYC, p53, p21 and BCL2 (n = 3) (b) and plated in semi-solid recipient mice transplanted with LSL-BcrBCR-ABL1 × Mb1-cre B cells transduced methylcellulose (c). Scale bars, 7 mm (c, top row), 2.5 mm (c, bottom row). d, with empty vector, IFITM3 or IFITM3(Y20E) (n = 7). i, The engraftment and Kaplan–Meier analyses of NOD-SCID-gamma (NSG) recipient mice injected expansion of luciferase-labelled leukaemia cells were monitored by luciferase with the indicated numbers of Ifitm3+/+ and Ifitm3−/− BCR-ABL1 (left) or NRASG12D bioimaging at the times indicated. j, Effects of IFITM3 or IFITM3(Y20E) on (right) B-ALL cells (n = 5). e, Frequencies of leukaemia-initiating cells (LIC) oncogenic signalling in LSL-BcrBCR-ABL1 × Mb1-cre B cell precursors measured by estimated with extreme limiting dilution analysis (ELDA) (90% confidence western blot and compared to empty vector. Phosphorylation of CD19 at Y531 interval; likelihood ratio test). f, Jeko1 MCL cells expressing doxycycline (CD19-pY531), of SRC at Y416 (SRC-pY416) and AKT at S473 were examined. Gel (Dox)-inducible Cas9 were transduced with IFITM3-targeting or non-targeting source data for a–c, f, g, j are shown in Supplementary Fig. 1. In c, g, two-tailed (NT) single-guide RNAs. Enrichment or depletion of targeted cells t-test. (Cas9+gRNA+) was monitored by flow cytometry upon treatment with

(Fig. 3h, i). Consistent with endosomal BCR functions22, interactions between BCR and IFITM3 occasionally colocalized with LAMP1+ endoso- IFITM3–PIP3 binding is needed to form lipid rafts mal compartments (Extended Data Fig. 6e). Therefore, BCR-mediated Reduced expression of integrins and adhesion receptors was asso- activation of SRC-mediated phosphorylation of Y20 of IFITM3 induces ciated with decreased homotypic adhesion of Ifitm3−/− pre-B cells, the accumulation of IFITM3 at the plasma membrane, where IFITM3 whereas membrane-bound IFITM3(Y20E) triggered the formation functions as a scaffold for CD19 and LYN in proximity to BCR molecules of large clusters (Extended Data Fig. 7). Because IKZF1 negatively to enable processive signal amplification (Fig. 3j). In resting B cells, regulates adhesion24,25, we examined whether this regulation is medi- IgM BCRs are excluded from lipid rafts and recruited upon encounters ated by transcriptional repression of Ifitm316. Overexpression of a with antigen23. By contrast, IgD BCRs are already in close proximity to dominant-negative IKZF1-mutant (termed IK6) markedly increased col- CD19 within lipid rafts in resting B cells, which suggests that IFITM3 ony formation, adhesion to stroma and surface expression of adhesion may specifically enable IgM BCRs. receptors in the presence—but not in the absence—of IFITM3. Although

494 | Nature | Vol 588 | 17 December 2020 a b d e B-ALL cells (PDX2) anti-HA PI3K signalling B-ALL cells (PDX2) anti-HA MCL cells (Jeko) anti-HA 4 NES = 1.9 PAK1-S174 q = 0.003 BirA BirA–IFITM3(Y20E) BirA BirA–IFITM3(Y20E) EV IFITM3(Y20E) BANK1-S12 2 ITGB1 PTPRC CXCR4-S321 CD44 DOCK1 SPN-S363/S365 CXCR4 ITGB1 TNS1-S1314 0 CD22 MS4A1 CD19-S480 ROCK1 BLNK RPS6-S236 BCR signalling DOCK1 CD72 FAK-S392/S394 PIK3R4 FAK PHIP-S879 NES = 1.6 1.0 RICTOR SYK PAG1-S50/S61 q = 0.02 BTK CXCR4 CD19-Y531 BLNK CD19 CD19-S353 AKT2 PIKFYVE MS4A1-S231 0 SYK PLCG2 CD19-S497/S494 ITGA5 PIK3C2B PIK3R4-S853 PIK3C3 ITGA5 -transformed fold change Integrin receptor GAB1 RPS6KB1-S427 2 CTNND1 RICTOR-S1385 PIKFYVE IRS2 log 2 FAK ROCK1 CD19-Y500 PIP5K1 PIK3CA BLNK-S219 0 CD19 AKT2 CD19-T356 CD72 CD19-T358 NES = 1.7 PLCG2 –2 q = 0.007 Proteinabundance (z score) Proteinabundance (z score): BCR signalling Integrin PI3K signalling –2 02 –2 02 chEV IFITM3 Y20E PLA signals per cell i PLA CD79B–IFITM3 Membrane stain WGA –+–+–+Anti-HA n = 250; 380; 405; 85 SRC-pY416 2 LYN CD19-pY531 20 CD19 P = 0.000 AKT-pS473 10 AKT

β-Actin A signals per cell 0 PL e f WCL IP: Flag 0 mi n 3 mi n Isotype 0 min3 min30 min anti-IgM 30 min Isot yp 3 3 g WCL IP:Flag FITM FITM EV I EV I 3 3

CD19 EV IFITM EV IFITM LYN Integrin-β1 j BCR BCR SYK CD19 CD19 CXCR4 PI3KCδ FAK STAT5 STAT5- PAK1 pY694 AKT Flag PIP3 IFITM3 PIP3 Flag LYN eIF4E eIF4E LYN

Fig. 3 | IFITM3 links components of the BCR and integrin receptor pathways and eIF4E as specificity controls. WCL, whole-cell lysates. h, i, Proximity to PI3K signalling. a, Levels of proteins phosphorylated at the indicated ligation assay (PLA) in Jeko1 cells upon BCR stimulation, assessed for proximity residues in PDX2 B-ALL cells transduced with HA-tagged IFITM3(Y20E) or of CD79B to IFITM3. Representative images with PLA signal (red), nuclei (DAPI) empty vector (EV) control were identified by mass spectrometry upon IFITM3 and plasma membrane (WGA) (green). Scale bars, 5 μm (i). h, Quantification of crosslinking with anti-HA antibodies. b, Feature set enrichment analysis (FSEA) PLA signals per cell (two-tailed t-test). j, Structural model of IFITM3-mediated for phosphorylated proteins in the BCR (red), integrin (blue) and PI3K (grey) signal amplification between CD19 and LYN. In resting B cells (left), IFITM3 is pathways, ranked by log2-transformed fold change (Kolmogorov–Smirnov). localized in endosomes. Upon antigen encounter (right), LYN-mediated c, Levels of SRC-pY416, LYN, CD19-pY531, CD19, AKT-pS473 and AKT assessed in phosphorylation of IFITM3 induces membrane translocation, acting as a ICN12 cells expressing IFITM3–HA, IFITM3(Y20E)–HA or HA-tagged empty scaffold for CD19 and LYN in proximity to BCR molecules. BCR–CD19–IFITM3 vector upon IFITM3 crosslinking. d, e, Interactomes of BirA–IFITM3(Y20E) or complexes form clusters for PI3K activation and accumulation of PIP3 in lipid empty vector control expressed in PDX2 B-ALL (d) and Jeko1 MCL (e) cells. f, g, rafts. In a–i, n = 3. For gel source data, see Supplementary Fig. 1. In a, d, e, Validation of IFITM3-interacting proteins by anti-Flag co-immunoprecipitation row-scaled protein abundance ranked by fold change; key proteins of interest (IP) in PDX2 cells transduced with Flag–IFITM3 or Flag-tagged empty vector, are highlighted. followed by western blotting for validation of interacting proteins, and STAT5 dominant-negative inhibition of IKZF1 relieved transcriptional repres- to PIP3, but not to any of the other 14 lipids (Fig. 4d). Because IFITM3 sion of adhesion receptors, the normal expression and function of function is required to retain normal PIP3 levels, we tested whether these receptors at the cell surface still required IFITM3 and its ability exogenous delivery of PIP3 could rescue Ifitm3 deficiency in mice. to link integrins and adhesion receptors to the PI3K pathway. Loss of Insertion of exogenous PIP3 into the cell membrane partially restored IFITM3, and reduced activity of the SRC kinase LYN and CD19–PI3K sig- AKT and MYC activation and colony formation, but not CD19 and SRC nalling, could broadly affect lipid rafts and short-circuit BCR signalling. phosphorylation or surface expression of lipid-raft-associated recep- Consistent with this scenario, the lipid-raft components cholesterol and tors (Extended Data Fig. 8b–d). Therefore, IFITM3 promotes PI3K sig- ganglioside GM1 were markedly reduced in Ifitm3−/− B cells (Fig. 4a). In nalling not only by increasing the amount of PIP3 but also by integrating addition, BCR engagement increased membrane rigidity in IFITM3+/+ PIP3 into signalling complexes in lipid rafts. MCL cells. However, membrane stiffening in response to BCR engage- ment was significantly reduced in IFITM3−/− MCL cells (Extended Data Fig. 8a). Consistent with defects in PI3K signalling, the ratios of PIP3 to Structural basis of IFITM3 binding to PIP3 the PI3K substrate PIP2 were reduced substantially in mouse Ifitm3−/− Proteins that contain a cluster of four or more basic residues located at B cells and patient-derived IFITM3−/− B-ALL cells. By contrast, overex- the membrane–solution interface can laterally sequester PIP3, which pression of IFITM3 and IFITM3(Y20E) increased PIP3:PIP2 ratios by does not require a PH domain or any other specific structure26. To map about 3-fold and 5.5-fold, respectively (Fig. 4b, c). To elucidate how PIP3 binding to specific portions of IFITM3, we repeated lipid-binding IFITM3 interacts with plasma membrane lipids, we probed 15 distinct assays with biotin-tagged fragments, including the N terminus, the lipid classes for binding to IFITM3 in vitro. IFITM3 bound selectively first intramembrane α-helix and the conserved intracellular loop. PIP3

Nature | Vol 588 | 17 December 2020 | 495 Article abce

Isotype No 6 600 80

l lipin l e

120 120 –5 c 60 +/+ B Ifitm3 P = 0.001 -s 400 P = 0.0001 40 Ifitm3/ P = 0.028 Pre 80 80 20 P = 6.7 × 10

Control IFITM3–biotin to biotin PIP3/PIP2 (%)

L 0 40 40 200 P = 0.0004 AL PI4P PIP2 PIP3 PIP2 PIP3 PIP2 PIP3 PIP2 PIP3 PIP2 PIP3 PI4P PI4P PI4P PI4P B- Ifitm3+/+ Ratio of Relative Ifitm3/ 0 0 0 1–57 58–70 71–105 71–88 89–105 GangliosideGM1 Filipi nIII N terminus IM-α CIL 71–105 TM-α 1 Y20 58 70 105 133 d T S IFITM3-CIL mutations

G GST

2 o Y20

t 200 IFITM3–GST o

i K83

t 100 GST a R85

R IFITM3 R87 f Biotin 60 IFITM3 K88 biotin 40 R85A K104 to 20 R85A/R87A/K88A j –+–+–+–+–+–+–+ Anti-HA 0 K83A/K104A AKT-pS473 Ratio Biotin IFITM3 g AKT R85A S6K-pT389 R85A/R87A/K88A S6K- K83A/K104A pT421/S424 2 3 n e e er ho PI3K signallin P PA

Ins S6K elin Etn - - -S (4)P -C DAG

ontrol -Actin cerid β lycerol (4,5) Ptd Ptd Ptd Ptd -g ec Sulfatid (3,4,5)P igly – + – + – + – + – + – + – + Anti-HA Cardiolipi PtdIns Cholesterol Tr Ptd k

PtdIns CD19-pY531 Sphingomy PtdIns Negativ CD19 g PIP3 IFITM3 PIP3 SRC-pY416

LYN BCR signalling K88 K104 β-Actin

+ + + + + + + Anti-HA R87        l PAK1/3- pS199/204 R85 PAK2- pS192/197 K83 PAK1 FAK- h n = 200; 250; 2,500; 2,500 i receptors pY576/577 )

K83 R85 K83A R85A IFITM3 K83A R85A –1 FAK-pY397 0 K83 80 FAK R85 60 CXCR4- –200 40

R87 and adhesion pS339 20 K88 CXCR4

–400 P = 3.2 × 10 0 K104 Integrin- 1 Integrin β –600 R85and R87 R85, R87 and K88 -Actin –800 β

–190 K83 and K104 Interaction energy (kJ mol

Fig. 4 | Structural basis of IFITM3-mediated regulation of PIP3 signalling within 3.5 Å. h, Interaction energies of PIP3 with basic residues K83 or R85 from lipid rafts. a, Ganglioside GM1 and cholesterol (stained with filipin III) averaged over molecular dynamics simulation trajectories (median levels levels measured in Ifitm3+/+ and Ifitm3−/− pre-B (top) and B-ALL (bottom) cells. indicated by red dotted line) (Methods). i, Binding affinity for PIP3 as shown as b, c, Ratios of PIP3 and PIP2 were measured in Ifitm3+/+ and Ifitm3−/− mouse a heat map of contact frequencies between PIP3 and each residue. j–l, (b, left), and human B-ALL cells (PDX2) (b, right), and upon expression of empty Levels of AKT-pS473, AKT, S6K phosphorylated at T389 (S6K-pT389), S6K vector, IFITM3 or IFITM3(Y20E) (c). d, In vitro lipid-binding assays for phosphorylated at T421 and S424 (S6K-pT421/S424), CD19-pY531, CD19, glutathione-S-transferase-tagged IFITM3 (GST–IFITM3) and 15 lipid classes. SRC-pY416, LYN, PAK1 and PAK3 phosphorylated at S199 and S204 Recombinant GST tag was used as baseline. e, Lipid-binding assays were (PAK1/3-pS199/204), PAK2 phosphorylated at S192 and S197 (PAK2-pS192/197), performed for biotin–IFITM3 N terminus (amino acids 1–57), the first PAK1, FAK phosphorylated at Y397 (FAK-pY397), FAK phosphorylated at Y576 intramembrane α-helix (IM-α) (amino acids 58–70), conserved intracellular and Y577 (FAK-pY576/577), FAK, CXCR4 phosphorylated at S339 loop (CIL) (amino acids 71–105) and transmembrane α-helix (TM-α) (amino (CXCR4-pS339), CXCR4 and integrin-β1 in PDX2 cells expressing empty vector, acids 89–105). f, Lipid-binding assays to study interactions of PIP3 with IFITM3–HA, IFITM3(Y20E)–HA, and the R85A, K83A, R85A/R87A/K88A and biotinylated fragments of the IFITM3 conserved intracellular loop that carry K83A/K104A mutants of IFITM3, upon IFITM3 crosslinking with anti-HA mutations of basic residues. g–i, PIP3 binding to K83 and K104, and to R85, antibodies. In a–f, j–l, n = 3, For gel source data, see Supplementary Fig. 1. R87 and K88, basic residue patches (g). Dashed lines indicate PIP3 contacts In b, c, e, h, two-tailed t-test.

binding was detected only for the conserved intracellular loop (Fig. 4e), interaction with one single basic residue (R85) (−213 kJ mol−1), which was which contains five basic residues that are in close proximity in sequence energetically weaker than the two charge interactions with PIP3 at K83 and that spatially cluster into two distinct basic patches. To address why and K104 (−704 kJ mol−1) (P = 0.0008). Two-residue contacts were rare IFITM3 preferentially binds to PIP3 over PIP2, we performed multi-scale for PIP2 and more common for PIP3 (P = 0.011) (Extended Data Fig. 9). molecular dynamics simulations of IFITM3 in multi-component lipid To experimentally test the predicted function of the basic residues of bilayers that mimic the plasma membrane. All-atom molecular dynam- the conserved intracellular loop, we performed PIP3 binding assays for ics simulations revealed that the representative binding pose of PIP2 biotinylated fragments of the IFITM3 conserved intracellular loop that from its most populated conformation cluster showed a charge carried mutations of these residues. The R85A and R85A/R87A/K88A

496 | Nature | Vol 588 | 17 December 2020 mutants largely retained the ability to bind PIP3, which was abrogated 5. Zhao, X. et al. Identification of residues controlling restriction versus enhancing activities of IFITM proteins on entry of human coronaviruses. J. Virol. 92, e01535-17 for the K83A/K104A mutant (Fig. 4f). To understand the structural (2018). basis of this difference, we modelled the interaction of PIP3 with each 6. Jia, R. et al. The N-terminal region of IFITM3 modulates its antiviral activity by regulating of the mutants of the basic residues (K83A/K104A, R85A/R87A/K88A IFITM3 cellular localization. J. Virol. 86, 13697–13707 (2012). 7. Chesarino, N. M., McMichael, T. M., Hach, J. C. & Yount, J. S. Phosphorylation of the and R85A/R87A) using all-atom molecular dynamics simulations. antiviral protein interferon-inducible transmembrane protein 3 (IFITM3) dually regulates PIP3 shows an interaction energy that is three times stronger with its endocytosis and ubiquitination. J. Biol. Chem. 289, 11986–11992 (2014). K83 and K104 than with R85, R87 and K88 (Fig. 4g–i). PIP3 makes 8. Everitt, A. R. et al. IFITM3 restricts the morbidity and mortality associated with influenza. Nature 484, 519–523 (2012). dual-pronged salt-bridge interactions with K83 and K104, whereas 9. Makvandi-Nejad, S. et al. Lack of truncated IFITM3 transcripts in cells homozygous it interacts only with R85 and not with R87 and K88 in the other basic for the rs12252-C variant that is associated with severe influenza infection. J. Infect. Dis. patch. The R87 and K88 residues form intra-protein salt bridges 217, 257–262 (2018). 10. Kohn, A. Early interactions of viruses with cellular membranes. Adv. Virus Res. 24, with D92 and D86, respectively, and are not available for interaction 223–276 (1979). with PIP3. 11. Liu, S. Y., Sanchez, D. J. & Cheng, G. New developments in the induction and antiviral effectors of type I interferon. Curr. Opin. Immunol. 23, 57–64 (2011). 12. Li, D. et al. KLF4-mediated negative regulation of IFITM3 expression plays a critical role in colon cancer pathogenesis. Clin. Cancer Res. 17, 3558–3568 (2011). IFITM3 amplifies PI3K, integrin and BCR signalling 13. Zhang, D., Wang, H., He, H., Niu, H. & Li, Y. Interferon induced transmembrane protein 3 To experimentally validate this model, we globally identified changes regulates the growth and invasion of human lung adenocarcinoma. Thorac. Cancer 8, 337–343 (2017). in signal transduction that depend on K83 and R85 residues in human 14. Spence, J. S. et al. IFITM3 directly engages and shuttles incoming virus particles to B-ALL and MCL B cells. The most prominent differences were enrich- lysosomes. Nat. Chem. Biol. 15, 259–268 (2019). ments for phosphorylation sites associated with PI3K, BCR and adhe- 15. Harvey, R. C. et al. Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome- sion receptor signalling (Extended Data Fig. 10a, b). Although most wide DNA copy number alterations, clinical characteristics, and outcome. Blood 116, phosphorylation events induced by membrane-bound IFITM3(Y20E) 4874–4884 (2010). were lost upon mutation of K83, this was not the case for substitu- 16. Witkowski, M. T. et al. Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome. J. Exp. Med. 214, 773–791 (2017). tion of the R85 residue (Extended Data Fig. 10c, d). Proximity ligation 17. Fujimoto, M. et al. CD19 regulates Src family protein tyrosine kinase activation in B assays revealed IFITM3-dependent recruitment of multiple proteins lymphocytes through processive amplification. Immunity 13, 47–57 (2000). in the PI3K, BCR and integrin signalling pathways. However, almost 18. Okkenhaug, K. et al. Impaired B and T cell antigen receptor signaling in p110δ PI 3-kinase mutant mice. Science 297, 1031–1034 (2002). all of these interactions were lost upon mutation of K83 (Extended 19. Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in Data Fig. 10e–h). Biochemical studies revealed that crosslinking of mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39–50 IFITM3(Y20E) resulted in massive activation of PI3K and integrin recep- (1995). 20. Chan, L. N. et al. Metabolic gatekeeper function of B-lymphoid transcription factors. tor pathways, whereas CD19 and SRC were constitutively phosphoryl- Nature 542, 479–483 (2017). ated in the presence of IFITM3(Y20E) (Fig. 4j–l). The R85A mutation 21. Wang, Y. et al. The physiologic role of CD19 cytoplasmic tyrosines. Immunity 17, 501–514 had only minor effects, whereas the substitution of K83 almost entirely (2002). 22. Phelan, J. D. et al. A multiprotein supercomplex controlling oncogenic signalling in abolished IFITM3-dependent signal transduction. In contrast to the lymphoma. Nature 560, 387–391 (2018). triple-mutant IFITM3(R85A/R87A/K88A), the IFITM3(K83A/K104A) 23. Kläsener, K., Maity, P. C., Hobeika, E., Yang, J. & Reth, M. B cell activation involves nanoscale double mutant lost all activity in PI3K, BCR and adhesion receptor receptor reorganizations and inside-out signaling by Syk. eLife 3, e02069 (2014). 24. Schwickert, T. A. et al. Stage-specific control of early B cell development by the pathways (Fig. 4j–l). transcription factor Ikaros. Nat. Immunol. 15, 283–293 (2014). In summary, we have revealed a function for IFITM3 as a PIP3 25. Joshi, I. et al. Loss of Ikaros DNA-binding function confers integrin-dependent survival on scaffold in the amplification of PI3K signalling. In Ifitm3−/− B cells, pre-B cells and progression to acute lymphoblastic leukemia. Nat. Immunol. 15, 294–304 (2014). lipid rafts were depleted of PIP3, which resulted in marked defects 26. McLaughlin, S. & Murray, D. Plasma membrane phosphoinositide organization by protein in lipid-raft-associated BCR and integrin receptor signalling. electrostatics. Nature 438, 605–611 (2005). IFITM3-dependent amplification of PI3K signalling, acting—in part— Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in downstream of the BCR and adhesion receptors, is critical for the published maps and institutional affiliations. rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent © The Author(s), under exclusive licence to Springer Nature Limited 2020, corrected publication 2021 signalling complexes and amplify PI3K signalling for malignant trans- formation. 1Center of Molecular and Cellular Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA. 2Department of Computational and Quantitative Medicine, City of Hope Comprehensive Cancer Center, Duarte, CA, USA. 3Department of Systems Biology, City of Online content Hope Comprehensive Cancer Center, Duarte, CA, USA. 4Department of Laboratory Medicine, 5 Any methods, additional references, Nature Research reporting sum- University of California San Francisco, San Francisco, CA, USA. Koch Institute for Integrative 6 maries, source data, extended data, supplementary information, Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, acknowledgements, peer review information; details of author con- UK. 7Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia tributions and competing interests; and statements of data and code and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. availability are available at https://doi.org/10.1038/s41586-020-2884-6. 8Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL, USA. 9Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. 10Dana Farber Cancer Institute, Boston, MA, USA. 11Harvard Medical School, Boston, 1. Bailey, C. C., Zhong, G., Huang, I. C. & Farzan, M. IFITM-family proteins: the cell’s first line MA, USA. 12Department of Biological Engineering, Massachusetts Institute of Technology, of antiviral defense. Annu. Rev. Virol. 1, 261–283 (2014). Cambridge, MA, USA. 13Department of Medicine, Washington University School of Medicine 2. Diamond, M. S. & Farzan, M. The broad-spectrum antiviral functions of IFIT and IFITM in St Louis, St Louis, MO, USA. 14Department of Molecular Microbiology, Washington proteins. Nat. Rev. Immunol. 13, 46–57 (2013). 15 3. Brass, A. L. et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, University School of Medicine in St Louis, St Louis, MO, USA. Department of Pathology and West Nile virus, and dengue virus. Cell 139, 1243–1254 (2009). Immunology, Washington University School of Medicine in St Louis, St Louis, MO, USA. 4. Zhang, Y. et al. Interferon-induced transmembrane protein 3 genetic variant rs12252-C 16Department of Immunobiology, Yale University, New Haven, CT, USA. ✉e-mail: markus. associated with disease severity in coronavirus disease 2019. J. Infect. Dis. 222, 34–37 (2020). [email protected]

Nature | Vol 588 | 17 December 2020 | 497 Article Methods pre-leukaemic B cell precursors expressing BCR–ABL1, LSL-Bcr+/BCR-ABL1 mice33 were crossed with Mb1-cre strain (Mb1-cre × LSL-Bcr+/ BCR-ABL1) for No statistical methods were used to predetermine sample size. excision of a stop-cassette in early pre-B cells. For in vivo oncogenic The experiments were not randomized and investigators were not priming assay with Mb1-cre × LSL-Bcr+/ BCR-ABL1 B cell precursors, 8- to blinded to allocation during experiments and outcome assessment. 10-week-old female NSG mice were randomly allocated before injec- tion. Temperatures of 18–23 °C with 40–60% humidity were maintained Analysis of patient gene expression and outcome data with 14-h light/10-h dark cycle. The following scores were considered as Gene expression microarray data from three large cohorts of patients end-point: (1) a failure to eat food or drink water for 24 h; (2) a failure to with pre-B ALL were downloaded from GSE531427 (the Eastern Coopera- make normal postural adjustments or to display normal behaviour; and tive Oncology Group (ECOG) Clinical Trial E2993), GSE1187728 (the Chil- (3) when tumour burden reached 1.5 cm × 1.5 cm × 1.5 cm (tumour ulcera- dren’s Oncology Group (COG) Clinical Trial P9906), St. Jude Research tion was not expected). If a mouse lost 25% of its initial body weight (or Hospital paediatric ALL29 (http://www.stjuderesearch.org/site/data/ reached 16 g of body weight, regardless of the initial weight), or if we ALL3/). In ECOG E2993, pretreatment bone marrow or peripheral observed a weight loss of 15% on 2 sequential weight measurements, blood samples were obtained at diagnosis before any treatment from we euthanized the mouse immediately. All mouse experiments were 83 patients with Ph+ B-ALL enrolled in the Medical Research Council subject to institutional approval by the Beckman Research Institute of (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) E2993 City of Hope Animal Care and Use Committee. phase III trial. In data set from St. Jude childhood, 15 cases of Ph+ B-ALL were selected from the original 327 diagnostic bone marrow aspirates. Mouse primary and leukaemia cells Minimal residual disease (MRD) data were available for patients with Bone marrow cells from 6–10-week-old mice were collected by flushing paediatric high-risk B-ALL (COG P9906). IFITM3 expression levels were cavities of the femur and tibia with chilled PBS, followed by filtering measured in patients with either a MRD positive (MRD+) status or a through 40-μm strainer to yield a single-cell suspension. Spleen or MRD negative (MRD-) status. In COG P9906 clinical outcome, expres- thymus cells were directly extracted by forcing tissues through a 40-μm sion profiles were obtained in pretreatment leukaemic samples from strainer into chilled PBS. Filtered cells were further incubated with 207 uniformly treated children with high-risk ALL30, a risk category lysis buffer (RBC Lysis Buffer, BioLegend) to lyse erythrocytes. After largely defined by pretreatment clinical characteristics. Patients had washing with PBS, cells were subjected to further experiments. For MRD tested by flow cytometry with two combinations (CD20/CD10/ IL-7-dependent pre-B cell culture, bone marrow cells were collected CD45 or CD9/CD19/CD34/CD45), and were defined as MRD positive or and cultured in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO) MRD negative at the end of induction therapy (day 29) using a thresh- with GlutaMAX containing 20% FBS, 50 μM 2-mercaptoethanol, 100 old of 0.01% as previously described31. Then, RNA was purified from IU ml−1 penicillin, 100 μg ml−1 streptomycin in the presence of 10 ng 207 pretreatment diagnostic samples with more than 80% blasts (131 ml−1 recombinant mouse IL-7 (Peprotech). For the BCR–ABL1 leukae- bone marrow, 76 peripheral blood) and subjected to microarrays. mia model, pre-B cells were retrovirally transformed with BCR-ABL1 Log-rank test was used to assess statistical significance. and then IL-7 was removed to select the transduced cells. NrasG12D ALL cells were selected with puromycin (GIBCO) and were maintained with Primary human samples and cell lines IL-7-supplemented IMDM. Patient samples (Supplementary Table 7) were sourced ethically from patients who gave informed consent, and were in compliance with the Retroviral and lentiviral transduction internal review boards of the Beckman Research Institute of City of Retroviral supernatant was generated by cotransfection of HEK 293FT Hope. We have complied with all relevant ethical regulations. Patient cells with retroviral constructs together with pHIT60 (gag-pol) and samples were harvested from biopsy of bone marrow from patients pHIT123 (for mouse) or pHIT456 (for human ecotropic envelope) with ALL at the time of diagnosis or relapse. All samples were trans- using Lipofectamine 2000 (Invitrogen). Lentiviral supernatant for planted into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice CRISPR-mediated gene editing was produced by cotransfection of (NSG mice, The Jackson Laboratory) through tail-vein injection. After HEK 293FT cells with lentiviral constructs together with pCDNL/BH samples were collected, patient-derived primary human pre-B ALL and EM140. Transfected HEK 293FT cells were cultured in high-glucose xenografts were cultured on OP9 stroma in Alpha Minimum Essential Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) with GlutaMAX Medium (MEMα; Life Technologies) with GlutaMAX containing 20% containing 10% fetal bovine serum, 100 IU ml−1 penicillin, 100 μg ml1 fetal bovine serum (FBS), 100 IU ml-1 penicillin, 100 μg ml-1 streptomy- streptomycin, 25 mmol l−1 HEPES, 1 mmol l−1 sodium pyruvate and 0.1 cin and 1 mM sodium pyruvate. The human cell lines (Supplementary mmol l−1 non-essential amino acids for 16 h. After sodium butyrate Table 8) were cultured in RPMI-1640 (GIBCO) with GlutaMAX contain- (10 mM) induction for 8 h, the virus supernatant was collected and ing 20%FBS, 100 IU ml-1 penicillin, 100 μg ml-1 streptomycin at 37 °C filtered through a 0.45-μm filter. The virus-containing supernatants in a humidified incubator with 5% CO2. All human primary samples were loaded by centrifugation (2,000g, 90 min at 32 °C) on 50 μg and cell lines were tested negative for mycoplasma by detection kit ml−1 retronectin (Takara)-coated non-tissue culture 6-well plates. For (MycoAlert PLUS, LONZA). retroviral transduction, 3–5 × 106 cells were transduced per well by centrifugation at 600g for 30 min in the appropriate culture medium

Genetic mouse models and maintained at 37 °C at 5% CO2 for 48 h. For lentiviral transduction, Genetic mouse models used in this study are listed in Supplementary 3–5 × 106 cells per well were centrifuged at 600g for 30 min in the pres- scid tm1Wjl Table 9. NOD.Cg-Prkdc Il2rg /SzJ (NSG) mice were purchased ence of lentiviral supernatant and maintained at 37 °C at 5% CO2. The from Jackson Laboratory. Ifitm3tm1Masu (ref. 32) mice were backcrossed lentiviral supernatant was replaced with fresh medium the next day. to the C57BL/6J background for more than eight generations. B6.C(Cg)-Cd79atm1(cre)Reth/EhobJ (Mb1–Cre) mice were purchased from Western blotting Jackson Laboratory. B6.129S2-Ighmtm1Cgn/J (μMT) mice were purchased PBS-washed cells were lysed in CelLytic buffer (Sigma-Aldrich) sup- from Jackson Laboratory. C;129S4-Ptentm1Hwu/J (Ptenfl/fl) mice were pur- plemented with 1% protease inhibitor cocktail (Roche Diagnostics), chased from Jackson Laboratory. For mice bred in-house, littermates 1% phosphatase inhibitor cocktail (EMD Millipore) and 1 mM PMSF on of the same sex were randomized to experimental groups. For the ice. A total of 10 μg of cell lysates was separated on mini-precast gels in vivo leukaemia initiation assay, 8- to 10-week-old female NSG mice (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad). were randomly allocated before injection. To generate a model for Membranes were probed with the appropriate primary antibodies. Membranes were then incubated with alkaline-phosphatase conjugated secondary antibodies (Invitrogen) and chemiluminescent substrate Inducible reconstitution of CD19 (Invitrogen) and were further detected by film exposure, UVP Bio- For inducible CD19 reconstitution, mouse CD19 was fused to the Spectrum 810 Imaging System (Thermo Fisher Scientific) or by using ligand-binding domain of a mutant oestrogen receptor (ERT2) at the ChemiDoc MP Imaging System (BioRad). Antibodies used in this the C terminus of CD19. BCR–ABL1-transformed mouse B-lineage study are provided in Supplementary Table 10, and were used at 1:750 ALL cells were retrovirally transduced with MSCV CD19-ERT2-Puro. to 1:1,000 dilution in blocking buffer. Puromycin-resistant cells were treated with 4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich) or vehicle control to induce reconstitution Flow cytometry of CD19. PBS-washed cells were blocked with Fc blocker for 10 min on ice and then stained with the appropriate antibodies listed in Supple- Cell viability assay mentary Table 10, or with isotype control, for 25 min on ice. Cells One hundred thousand BCR–ABL1-transformed mouse B-lineage ALL were then washed and resuspended in chilled PBS containing 0.75 cells were seeded in a volume of 100 μl in medium in 1 well of a 96-well μg ml−1 of DAPI to exclude dead cells. Acquisition was performed by plate (BD Biosciences). Imatinib (LC Laboratories) was added at the LSRFortessa flow cytometer (BD Biosciences) with BD FACSDIVA indicated concentration in a total volume of 150 μl. After culturing for software. The fluorescence-based cell sorting was performed by 3 days, 15 μl of resazurin (R&D) was added into each well and incubated FACSAria II (BD Biosciences) with BD FACSDIVA software. FACS data for 4 h at 37 °C. Medium without cells was used as blank. The fluores- were analysed with FlowJo software (FlowJo). For apoptosis analyses, cence was read at 535 nm and the reference wavelength was 590 nm. annexin V and 7-AAD (BD Biosciences) were used. For cell cycle analy- Relative viability was calculated using baseline values of vehicle-treated sis, the BrdU flow cytometry kit (BD Biosciences) was used according cells as a reference. to manufacturer’s instructions. For intracellular staining of cytoplas- mic proteins, cells were first stained for cell surface antigens and Colony-forming assay subsequently fixed in fixation and permeabilization solution (BD The methylcellulose colony-forming assays were performed with Biosciences) containing 4% paraformaldehyde and the detergent 10,000 cells. Cells were resuspended in mouse MethoCult medium saponin. Cells were then washed and resuspended in Perm/Wash (without cytokines for BCR-ABL1-transformed cells; with IL-7 for Buffer (BD Biosciences) and stained with the appropriate antibodies. NRASG12D-expressing cells) and cultured on 3-cm-diameter dishes, For statistical quantification, data were plotted with GraphPad Prism with an extra water supply dish to prevent evaporation. Colonies were 7 or SigmaPlot. FACS antibodies, listed in Supplementary Table 10, imaged and counted after seven days using GelCount (Oxford Optro- were pre-diluted 1:5–1:10. Two μl of diluted antibody was added to nix), Olympus IX71 microscope and Q-Capture pro 7. 1–2 million cells per 50 μl in PBS for final dilution of 1:50 for human cells or 1:200 for mouse cells. In vitro IFITM3–HA crosslinking Eight million patient-derived B-ALL cells per sample were resuspended Pharmacological inhibitors and reagents into complete medium and treated with either 2.5 μg ml−1 polyclonal Imatinib was purchased from LC Laboratories. Stock solutions were anti-HA (Abcam) or isotype control for the indicated times. F(ab) frag- prepared in sterile water at 10 mmol l−1 and administered at 10 μmol ments for the anti-HA antibody were generated by using the F(ab) l−1. Dasatinib was purchased from SelleckChem. Stock solutions were preparation kit following the manufacturer’s instructions (Thermo prepared in sterile DMSO at 25 μmol l−1 and administered at 25 nmol l−1 Fisher Scientific). F(ab) fragmentation was confirmed by Criterion (Supplementary Table 11). TGX Stain-Free Precast Gels (Bio-Rad).

Analysis of chromatin immunoprecipitation with sequencing Measurement of intracellular calcium mobilization data In Extended Data Fig. 2f, 1 million fresh splenocytes were incubated with IKZF1 chromatin immunoprecipitation with sequencing (ChIP-seq) the 4 μM Rhod-2-AM Ca2+-binding dye (Thermo Fisher Scientific) for 15 was performed as previously described34 with a patient-derived min at room temperature in dark. Cells were then resuspended in PBS human B-ALL xenograft cell line (LAX2), which expresses wild-type and maintained at 37 °C, and Ca2+ response was induced by adding 10 μg full-length IKZF1 and no detectable level of the dominant-negative ml−1 of polyclonal anti-IgM (Southern Biotech) at 50 s after acquisition IKZF1 isoform. ChIP-seq tracks (GSE58825) for the IKZF1 antibody in of background fluorescence. Intracellular Ca2+ mobilization in response LAX2 on IFITM3 gene promoter regions are shown in the bottom of to crosslinked IgM was measured up to 300 s by flow cytometry. Extended Data Fig. 1m. The y axis represents the normalized number of In Extended Data Fig. 4c, 1 × 106 viable Jurkat cells were incubated with reads per million reads for peak summit for each track. ChIP-seq tracks the 4 μM Fluo4-AM Ca2+-binding dye (Thermo Fisher Scientific) for (GSE86897) for the enrichment of RNAPII and H3K4me3 at the Ifitm3 15 min at room temperature in dark. Cells were then resuspended in PBS locus in pre-B cells from Ikzf1exon5fl/fl mice upon Cre-mediated deletion and maintained at 37 °C, and Ca2+ response was induced by adding 10 of Ikzf1 are shown in the top of Extended Data Fig. 1m. The ChIP-seq μg ml−1 of monoclonal (OKT3) purified NA/LE anti-human CD3 (Bioleg- peaks were called by the MACS peak-caller by comparing read density end) at 50 s after acquisition of background fluorescence. Intracellular in the ChIP experiment relative to the input chromatin control reads, Ca2+ mobilization in response to crosslinked IFITM3 was measured up and are shown as bars under each wiggle track. Gene models are shown to 300 s by flow cytometry. The same procedure was performed for in UCSC genome browser hg19. Integrative Genomics Viewer (IGV) was Extended Data Fig. 6f; 1 × 106 viable Jeko1 cells were incubated with the used to visualize ChIP-seq tracks. 4 μM Fluo4-AM (Thermo Fisher Scientific). Ca2+ response was induced −1 by adding 10 μg ml of polyclonal F(ab′)2 anti-human μ chain (Jackson Inducible expression of IKZF1 Immunoresearch). Human Philadelphia chromosome-positive (Ph+) pre-B ALL cells (BV173) carrying deletions of IKZF1 were transduced with Homotypic aggregation assay pRetroX-Tet3G-Neo. Neomycin-resistant cells were transduced with Ifitm3−/− B-ALL cells expressing C-terminal HA-tagged wild-type IFITM3, pRetroX-TRE3G-wild-type IKZF1-Puro. Expression of wild-type IKZF1 IFITM3 mutant vector (Y20E) or an empty vector were incubated with in puromycin-resistant cells was initiated by treatment with 1 μg ml−1 2.5 μg ml−1 of monoclonal (1D3) purified LEAF anti-mouse CD19 (Bio- of doxycycline (TetOn). legend) or 2.5 μg ml−1 polyclonal anti-HA (Abcam) or isotype controls Article at 37 °C at 5% CO2. After culturing for 24 h, homotypic aggregation was (RA3-6B2) anti-CD45R (B220, BD Biosciences), monoclonal anti-mouse visualized using light microscopy. CD3 (17A2, BioLegend) antibodies and biotinylated peanut agglutinin (B-1075, Vector Laboratories) for 45 min. Sections were washed and fur- In vivo oncogenic priming assay with Mb1-cre;LSL-Bcr+/BCR-ABL1 ther stained with Alexa Fluor 647 streptavidin (BioLegend) antibody for B cell precursors 45 min. All antibodies were diluted 1:100 to their original concentration To generate a model for pre-leukaemic B cell precursors expressing in blocking buffer. After washing with blocking buffer, sections were BCR–ABL1, BCR–ABL1 knock-in mice were crossed with Mb1–Cre strain mounted in ProLong Diamond Antifade Mountant (ThermoFisher Sci- (Mb1-cre × LSL-Bcr+/BCR-ABL1) for excision of a stop cassette in early pre-B entific). Images were acquired on a ZEISS LSL 880 confocal microscope cells. Bone marrow cells collected from Mb1-cre × LSL-Bcr+/BCR-ABL1 mice, and analysed on ZEN 2.3 (Zeiss) software. cultured in the presence of 10 ng ml−1 recombinant mouse IL-7 (Pepro- tech). IL-7 dependent pre-leukaemic B cells were labelled with retroviral ELISA measurements firefly luciferase and selected by blasticidin for 7 days. After selec- For determination of the concentrations of immunoglobulin isotypes tion, cells were further transduced with MSCV-IFITM3-HA-IRES-Puro, in sera, ELISAs were carried out according to the manufacturer’s pro- MSCV-IFITM3Y20E-HA-IRES-Puro or empty vector and selected by puro- tocol (Ig Isotyping Mouse Instant ELISA Kit for IgG1, IgG2b and IgM) mycin for 3 days.Viable cells (1 × 107) were injected via the tail vein into (Supplementary Table 11). NP-specific antibodies were measured by sublethally irradiated (200 cGy) NSG recipient mice. In vivo expansion ELISA using 10 μg/ml of NP(24)–BSA (Biosearch Technologies) as the and leukaemia burden were monitored by luciferase bioimaging (IVIS coating reagent. NP-specific IgM and IgG1 was detected using goat 100 bioluminescence and optical imaging system; Xenogen) at the anti–mouse IgM and IgG1 Fc-specific antibodies conjugated to horse- indicated time points. In brief, d-luciferin (Promega) dissolved in PBS radish peroxidase and developed with tetramethylbenzidine (Sigma). was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min Optical densities were determined by using an ELISA reader at 450 nm before measuring luminescence. All mice were anaesthetized by 5% (SpectraMax M3, Molecular Devices). isoflurane and continued during detection of light emission with 2% isoflurane introduced through a nose cone. A mouse was euthanized RNA-sequencing analysis when they showed signs of leukaemia burden such as a hunched back, Total mRNA from Ifitm3+/+ and Ifitm3−/− BCR-ABL1 or NRASG12D B-ALL cells weight loss and inability to move. Kaplan–Meier survival analysis was was extracted using RNeasy Kit (Qiagen) according to the manufac- performed using GraphPad Prism 7 (GraphPad Software) to compare turer’s instructions. Sequencing was performed on an Illumina Hiseq overall survival. Mantel–Cox log-rank test was used as statistical analy- 2500 (Illumina) instrument using the TruSeq SR Cluster Kit v.4-cBot-HS sis using GraphPad Prism 7 (Supplementary Table 4). (Illumina) with v.4 chemistry. Quality control of RNA-sequencing reads was performed using FastQC. For analysis, raw sequence reads were Cell adhesion assay mapped to the mouse genome (mm10) using STAR v.2.5.335, and the Ifitm3+/+ or Ifitm3−/− BCR-ABL1 B-ALL cells were transduced with frequency of genes was counted using featureCounts v.1.5.136. The MSCV-IK6-IRES-GFP or MSCV-IRES-GFP as negative control. GFP+ raw counts were then normalized using the trimmed mean of M values cells (1 × 105) sorted by FACS were cultured on 1.5 × 105 OP9 stroma method and compared using Bioconductor package ‘edgeR’37. Reads per on a 6-well plate with IMDM-GlutaMAX containing 20% FBS, 50 μM kilobase per million (RPKM) mapped reads were also calculated from 2-mercaptoethanol, 100 IU ml−1 penicillin and 100 μg ml−1 streptomy- the raw counts. For differential expression analysis, transcripts were cin. For calculation of the ratio of adherent cells to nonadherent cells, quantified using Salmon v.1.1.0 against gencode GRCm38 v.M24 tran- nonadherent cells were collected and counted with the trypan blue script annotations; normalization and statistical analysis were done in R exclusion method using the Countess II FL Automated Cell Counter using DESeq2 v.1.28.1. Differentially expressed genes were identified if (Life Technologies). After washing the plate with PBS twice, adherent RPKM ≥ 1 in at least one sample, fold change ≥ 2 and P ≤ 0.05 (Extended cells were detached with trypsinization and GFP+ B-ALL cells and were Data Fig. 3b). RPKM data were later used in the gene set enrichment counted with the trypan blue exclusion method using the Countess II FL analysis (GSEA). GSEA was performed using the DOSE package in R38;

Automated Cell Counter (Life Technologies) and ratios were calculated. genes were ranked by log2-transformed fold change; and gene sets were obtained from MSigDB or from internal data, as indicated. Adoptive transfer of purified B cells into μMT mice For the adoptive transfer of B cells, splenocytes from 7- to 10-week-old CRISPR-mediated gene deletion Ifitm3+/+ or Ifitm3−/− mice were negatively selected by MojoSort Mouse All lentiviral constructs expressing Cas9 nuclease and guide RNA were Pan B Cell Isolation Kit II (Miltenyi Biotec) using immunomagnetic purchased from Transomic Technologies. For gene deletion, cells were beads against CD3, CD4, CD8a, CD11c, CD49b, Ly6G/Ly6C (GR1) and transduced with pTOL-hCMV-Tet3G-Hygromycin. Hygromycin-resistant TER119. Ten million flow-sorted splenic B cells were intravenously cells were subsequently transduced with pCLIP-Tre3g-hCMV-Cas9- injected into μMT mice (B6.129S2-Ighmtm1Cgn/J) that lack the Cμ exon P2A-zsGreen. Expression of Cas9–P2A–zsGreen was induced at 1 μg and mature B cell development as a result of defective surface IgM ml−1 of doxycycline for 16 h, and zsGreen+ cells were sorted by flow expression. Reconstitution of donor B cells was determined by flow cytometry. Cells were washed out to remove doxycycline to turn off cytometry 20 days after injection. Recipient μMT mice were immunized Cas9 expression (TetOff) and then subsequently transduced with with the hapten NP coupled to a carrier protein (KLH) on day 0 and day pCLIP-gRNA-hCMV-RFP-gRNA. Sorted RFP+ cells were subjected to 7, and spleens were collected on day 12. further experiments. Gene deletion was initiated by treatment with 1 μg ml−1 of doxycycline (TetOn). Non-targeting guide RNA was used Immunization and immunohistology as control. Ifitm3+/+ and Ifitm3−/− littermate mice were immunized with 0.5 mg NP–KLH (Biosearch Technologies) in alum (Sigma-Aldrich) intraperi- Gene deletion by non-viral genome targeting toneally. Seven days later, mice were immunized a second time for five Chemically synthesized CRISPR RNAs (160 μM) and trans-activating days. Spleens were isolated at day 12 after immunization. The spleen was CRISPR RNAs (160 μM) were mixed 1:1 by volume and annealed by incu- embedded in optimum cutting temperature compound and 5-μm-thick bation at 37 °C for 30 min. Recombinantly produced Cas9 (40 μM) was cryosections were used for staining. Sections were fixed with acetone then mixed 1:1 by volume with gRNA to produce RNA ribonucleoprotein for 10 min, and nonspecific antigens were blocked in DPBS containing (RNP) complexes. RNPs were freshly complexed before electroporation. 2% FBS for 15 min. Sections were stained with 1:200 diluted polyclonal Electroporation was performed by using pulse code EH-115 on a Lonza 4D 96-well electroporation system. Predesigned Alt-R CRISPR–Cas9 eluted using a 150-min (IMAC) linear gradient of acetonitrile in 0.125% guide RNAs were purchased from IDT. Non-targeting control guide formic acid delivered at 280 nl/min. Tandem mass spectra (MS/MS) RNAs were purchased from IDT. were collected in a data-dependent manner with a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer using a top-twenty MS/MS Co-immunoprecipitation method, a dynamic repeat count of one and a repeat duration of 30 s. Co-immunoprecipitation was performed with the Pierce Crosslink Mag- Real-time recalibration of mass error was performed using lock mass netic IP/Co-IP kit according to manufacturer’s instructions (Thermo (Olsen) with a singly charged polysiloxane ion of m/z = 371.101237. MS/ Fisher Scientific). In brief, patient-derived Ph+ ALL cells (PDX2) were MS spectra were evaluated using SEQUEST and the Core platform from transduced with MSCV Flag-IRES-Puro or MSCV Flag-IFITM3-IRES-Puro Harvard University (by J. K. Eng, E. L. Huttlin and J. Villen). Files were and selected in puromycin for 3 days. Viable cells (5 × 107) were col- searched against the SwissProt Homo sapiens FASTA database. A mass lected and washed by PBS before lysis using IP lysis/Wash buffer accuracy of ±5 ppm was used for precursor ions and 0.02 Da for product (Thermo Fisher Scientific). Then, 5 μg of anti-Flag antibody M2 (F1804, ions. Enzyme specificity was limited to trypsin, with at least one tryptic Sigma-Aldrich) per sample was coupled to protein A/G magnetic beads (K- or R-containing) terminus required per peptide and up to four mis- and covalently crosslinked with 20 μM disuccinimidyl suberate. The cleavages allowed. Cysteine carboxamidomethylation was specified antibody crosslinked beads were incubated with cell lysate, washed to as a static modification, and oxidation of methionine and phospho- remove non-bound material and eluted in a low-pH elution buffer that rylation on serine, threonine and tyrosine residues were allowed as dissociates bound antigen from the antibody crosslinked beads. The variable modifications. Reverse decoy databases were included for enriched antigen in low pH was immediately neutralized and subjected all searches to estimate false discovery rates, and filtered using a 1% to western blotting. Jeko1 cells electroporated with either non-targeting false-discovery rate in the linear discriminant module of Core. Peptides RNP or IFITM3-targeting RNP complex were stimulated with 10 μg ml−1 were also manually filtered using a ±5ppm mass error range and pres-

F(ab′)2 fragment goat anti-human μ chain (Jackson Immunoresearch) ence of a phosphorylated residue. All quantitative results were gener- for the indicated time points at 37 °C and 5% CO2. Cells were immedi- ated using Skyline (MacLean) to extract the integrated peak area of the ately washed with chilled PBS and subjected to co-immunoprecipitation corresponding peptide assignments. Accuracy of quantitative data was with anti-CD19 antibody (no. 90176, CST). ensured by manual review in Skyline or in the ion chromatogram files.

PLA Bio-ID proteomics For PLA of pre-BCR or BCR–IFITM3, cells were incubated with 10 μg Patient-derived PDX2 B-ALL cells and Jeko1 MCL cells were transduced −1 ml F(ab′)2 fragment goat anti-human μ chain (Jackson Immunore- with Flag-tagged IFITM3(Y20E) or IFITM3(K83A/K104A) mutant con- search) for 5 min at 37 °C and 5% CO2. Cells were immediately washed structs with N-terminal Turbo-ID BirA (engineered biotin ligase) and with chilled PBS and subsequently fixed in fixation buffer (Biolegend) selected by puromycin for three days. Turbo-ID BirA-expressing con- containing 4% paraformaldehyde for 25 min on ice and then washed struct tagged with Flag was used as negative control. To induce bioti- with chilled PBS. For cellular membrane staining, cells were labelled nylation of proteins proximal to IFITM3, cells were treated with 50 with 5 μg ml−1 WGA conjugated to Alexa Fluor 488 (Thermo Fisher Sci- μmol l−1 biotin and with anti-HA antibody for simultaneous induction entific) for 5 min at room temperature. Cells were then permeabilized of IFITM3 crosslinking for 10 min. Cells were washed three times with in Perm/Wash Buffer (BD Biosciences) and then blocked in Duolink chilled PBS and lysed in IP/WASH buffer (Thermo Fisher Scientific) Blocking buffer for 30 min at room temperature. Cells were incubated with 1× HALT protease inhibitor (Thermo Fisher Scientific). The lysates with 1:150 diluted primary antibodies listed in Supplementary Table 10 were incubated by streptavidin C1 MyOne Dynabeads (Invitrogen) for overnight at 4 °C. For late endosome staining, cells were incubated 16 h at 4 °C. Unbound proteins were washed 3 times by 2% SDS–PBS, with anti-human LAMP1 conjugated to Alexa Fluor 488 (R&D Systems) 3 times by PBS and 3 times with pure water. The eluted proteins were with the primary antibodies. Cells were washed and settled on Cell-Tak gel-purified, followed by in-gel digestion and subjected to mass spec- (Corning)-coated Shandon Single Cytoslide by cytospin at 400g for 5 trometry. For liquid chromatography (LC)–MS/MS analysis, peptides min. PLA reactions were carried out according to the manufacturer’s were analysed using a Dionex UltiMate 3000 Rapid Separation LC protocol (Duolink, Sigma). In brief, primary antibodies were coupled system and a Orbitrap mass spectrometer (ThermoFisher Scientific). with Duolink in situ PLA PLUS or MINUS probes (Sigma-Aldrich), and Six-μl peptide samples were loaded onto the trap column, which was then probes were visualized with Duolink Detection Reagent Red 150 μm × 3 cm and in-house-packed with 3-μm C18 beads. The analytical (Sigma-Aldrich). Cells were mounted with Duolink in situ mounting column was a 75 μm × 10.5-cm PicoChip column packed with 3-μm C18 medium with DAPI (Sigma-Aldrich). Microscopy images were acquired beads (New Objectives). The flow rate was kept at 300 nl/min. Solvent using an Olympus IX3-55 and analysed by CellSens imaging software A was 0.1% formic acid (FA) in water and solvent B was 0.1% FA in ACN. (Olympus) and ImageJ. For quantification of PLA signals, one dot The peptide was separated on a 120-min analytical gradient from 5% was defined as pixel size of 5 × 5 by BlobFinder software. Statistical ACN and 0.1% FA to 40% ACN and 0.1% FA. The mass spectrometer was significance was calculated using unpaired Student’s t-test with Excel operated in data-dependent mode. The source voltage was 2.40 kV and and plotted with GraphPad Prism 7. the capillary temperature was 275 °C. MS1 scans were acquired from 400–2,000 m/z at 60,000 resolving power and automatic gain control Phosphoproteomic analysis (AGC) set to 1 × 106. The 15 most abundant precursor ions in each MS1 Patient-derived B-ALL (PDX2) cells transduced with HA-tagged scan were selected for fragmentation. Precursors were selected with IFITM3(Y20E) or empty vector control were incubated with 2.5 μg ml−1 of an isolation width of 1 Da and fragmented by collision-induced disso- polyclonal anti-HA (Abcam) for 5 min at 37 °C at 5% CO2. Cellular extracts ciation at 35% normalized collision energy in the ion trap; previously were prepared in urea lysis buffer, sonicated, centrifuged, reduced selected ions were dynamically excluded from reselection for 60 s. with DTT and alkylated with iodoacetamide. Fifteen mg total protein The MS2 AGC was set to 3 × 105. For data analysis, proteins were identi- for each sample was digested with trypsin, and 500 μg total protein for fied from the mass spectrometry raw files using Mascot search engine each sample was digested with LysC and trypsin mix for IMAC analysis. (Matrix science). MS/MS spectra were searched against the SwissProt Samples were purified over C18 columns and dried in a lyophilizer. human database. All searches included carbamidomethyl cysteine as Dried samples were resuspended and enriched with Fe-IMAC beads, a fixed modification and oxidized Met, deamidated Asn and Gln, and purified over C18 STAGE tips (Rappsilber). Replicate injections of each acetylated N terminus as variable modifications. Three missed tryptic sample were run nonsequentially on the instrument. Peptides were cleavages were allowed. The MS1 precursor mass tolerance was set to Article 10 ppm and the MS2 tolerance was set to 0.6 Da. A 1% false discovery rate peptide abundance and searched against the human Swiss-Prot anno- cut-off was applied at the peptide level. Only proteins with a minimum tated human proteome from Uniprot (downloaded with 20,303 of two peptides above the cut-off were considered for further study. For entries). The ‘match-between-runs’ option was selected to increase comparison to empty vector control, background peptide abundances peptide identifications and the ‘fast LFQ’ option was selected to for missing values were imputed from a Gaussian distribution centred calculate label-free quantification values of identified proteins. All around the minimum observed abundance using the MinProb method other settings were left at the default MaxQuant values. The MaxQuant from MSnbase package in R. output data were analysed using Perseus41 and the R program (ver- sion 3.4.0). Proteins annotated as ‘reverse’, ‘only identified by site’ and Cell-surface proteome analyses ‘potential contaminant’ were filtered out, as were proteins that were Cell-surface proteins were labelled with biotin using the N-linked quantified in less than two out of three biological replicates in at least glycosylation-site biotin labelling method39. In brief, 40 million Ifitm3+/+ one experimental group. Missing values were imputed on the basis or Ifitm3−/− B-ALL cells were washed twice and resuspended in 1 ml of of the normal distribution of the dataset as implemented by Perseus. ice-cold PBS, and treated with 1.6 mM sodium metaperiodate (VWR) Volcano plots were generated using output from a two-sample t-test at 4 °C for 20 min to oxidize the vicinal diols of sugar residues linked comparing the log2-transformed label-free quantification protein abun- to surface proteins. The cells were then washed twice in PBS to remove dance values from different cell lines with a false-discovery rate set to excess sodium metaperiodate. Cells were resuspended in 1ml of ice-cold 0.01. Validation was performed by flow cytometry (Supplementary PBS and treated with 1 mM biocytin hydrazide (Biotium) and 10 mM Table 1). aniline (Sigma-Aldrich) at 4 °C for 90 min with gentle mixing to bioti- nylate free aldehydes exposed on the sugar residues. After labelling, Cholesterol and lipid raft measurement cells were washed 3 times with ice-cold PBS to removed excess biotin, For the depletion of cholesterol, cells were preincubated with 5 mM frozen in liquid nitrogen and stored at −80 °C until further processing of methy-β-cyclodextrin for 30 min at 37 °C before filipin staining. for mass spectrometry. All experiments were performed in biological For ganglioside GM1 staining, cells were labelled with cholera toxin B triplicates with replicates collected from consecutive passages. Frozen using Vybrant lipid raft labelling kit (Molecular Probes), according to cell pellets were thawed on ice in 1 ml of RIPA buffer (Millipore) with the the manufacturer’s protocol. In brief, cells were labelled with cholera addition of 1× HALT protease inhibitors (Pierce). After incubation on toxin unit B conjugated with Alexa Fluor 594 on ice for 15 min, washed ice for 10 min, cells were disrupted by sonication and the lysates were twice with PBS. Cholera-toxin-B-labelled lipid rafts were crosslinked clarified by centrifugation at 17,000 rcf at 4 °C for 10 min. Clarified with anti-cholera-toxin-B antibody on ice for 15 min, washed twice with lysate was mixed with 500 μl of neutravidin agarose resin (Thermo PBS and analysed by flow cytometry. Fisher Scientific) and incubated at 4 °C for 2 h with end-over-end mix- ing. Neutravidin beads with captured biotinylated surface proteins In vivo transplantation of leukaemia cells were washed extensively by gravity flow to remove unbound proteins Mouse pre-B ALL cells transformed by BCR–ABL1 or NRAS(G12D) using 50 ml of 1× RIPA + 1 mM EDTA, followed by 50 ml of PBS + 1 M NaCl were injected into sublethally irradiated (200 cGy) NOD-scid IL2Rgnull and finally 50 ml of 50 mM ABC + 2 M urea buffer. Washed beads were (NSG) mice via the tail vein. Eight- to ten-week-old female NSG mice resuspended in digestion buffer (50 mM Tris pH 8.5, 10 mM TCEP, 20 mM were randomly allocated before injection. A mouse was euthanized 2-iodoacetamide and 1.6 M Urea) with 10 μg of added trypsin protease when they showed signs of leukaemia burden, such as a hunched back, (Pierce, 90057) to perform simultaneous disulfide reduction, alkyla- weight loss and inability to move, and then the bone marrow and/or tion and on-bead peptide digestion at room temperature overnight spleen were collected to test leukaemia infiltration by flow cytometry. (16–20 h). After digestion, the pH was dropped to about 2 with neat Kaplan–Meier survival analysis was performed using GraphPad Prism trifluoroacetic acid (Sigma-Aldrich) and the peptide mixture was 7 (GraphPad Software) to compare overall survival. The Mantel–Cox desalted using a SOLA-HRP column (Thermo Fisher Scientific) on a log-rank test was used as statistical analysis using GraphPad Prism 7 vacuum manifold. Desalted peptides were eluted with 50% acetoni- (Supplementary Table 3). The minimal number of mice in each group trile (Sigma-Aldrich) and 50% water with 0.1% TFA and dried down was calculated by using the ‘cpower’ function in Hmisc package of R. No completely in a speedvac. Dried peptides were resuspended in LC– blinding was used. All mouse experiments were subject to institutional MS grade water (Fisher) with 2% ACN and 0.1% FA. Peptide concentra- approval by the Beckman Research Institute of City of Hope Animal tion was measured using 280 nm absorbance on a Nanodrop, and the Care and Use Committee. peptide concentration was adjusted to 0.2 μg μl−1 for mass spectrom- etry runs. PIP3 quantification For Fig. 4, 60 million viable cells were resuspended in chilled 0.5 M LC–MS and data analysis for cell-surface proteome trichloroacetic acid (TCA) in a total volume of 1 ml and incubated on For each replicate, 1 μg of peptide was injected onto a Dionex Ultimate ice for 5 min. Cells were centrifuged at 3,000 rpm for 7 min at 4 °C and 3000 NanoRSLC instrument with a 15-cm Acclaim PEPMAP C18 (Thermo resuspended in 5% TCA and 1 mM EDTA in a total volume of 1 ml. After Fisher Scientific, 164534) reverse-phase column. The samples were vortexing for 30 s, cells were washed again with 5% TCA and 1 mM EDTA. separated on a 3.5-h nonlinear gradient using a mixture of buffer A (0.1% To extract neutral lipids, cells were resuspended in 1 ml of MeOH:CHCl3 FA) and B (80% ACN/0.1% FA), from 2.4% ACN to 32% ACN. Eluted pep- (2:1) and vortexed for 10 min at room temperature and centrifuged tides were analysed with a Thermo Q-Exactive Plus mass spectrometer. at 3,000 rpm for 5 min. After 1 more extraction of neutral lipids, the

The mass spectrometry survey scan was performed over a mass range acidic lipids were extracted by adding 750 μl MeOH:CHCl3:12 M HCl of 350–1500 m/z with a resolution of 70,000, with a maximum injection (80:40:1) with vigorous vortexing for 25 min at room temperature. time of 100 ms. We performed a data-dependent MS2 acquisition at a After centrifugation at 3,000 rpm for 5 min, supernatant was trans- 4 resolution of 17,500, AGC of 5 × 10 , and injection time of 150 ms. The ferred to a 2-ml centrifuge tube and mixed with 250 μl of CHCl3 and 15 most intense precursor ions were fragmented in the higher-energy 450 μl of 0.1 M HCl, vortexed for 30 s, and centrifuged at 3,000 rpm for collisional dissociation at a normalized collision energy of 27. Dynamic 5 min to separate organic and aqueous phases. Five hundred μl of the exclusion was set to 20 s to avoid over-sampling of highly abundant lower organic phases was collected into a clean 1.5-ml vial and dried in species. The raw spectral data files are available at the ProteomeX- a vacuum dryer for 1 h. Dried lipid was stored at −20 °C. On the day of change PRIDE repository (accession number PXD014691). Raw spectral assay, lipid samples were reconstituted with 200 μl of PBS containing 40 data were analysed using MaxQuant v.1.5.1.2 to identify and quantify 0.25% protein stabilizer; 20 μl was used for PI(4,5)P2 measurement and 50 the rest (180 μl) was used for PI(3,4,5)P3 measurement using an ELISA The reaction field coulomb was applied to describe electrostatics, kit (Echelon Biosciences) according to the manufacturer’s instructions. with cut-off at 1.1 nm. The cut-off for van der Waals interactions used was also 1.1 nm. We used the Leap-frog integrator, and a 20-fs time step for Exogenous delivery of PIP3 integration. All systems were solvated in coarse-grained water model Phosphatidylinositol 3,4,5-trisphosphate diC16 (Echelon Biosciences) adopted in the Martini forcefield water, neutralized with 0.15 M NaCl. was freshly reconstituted with PBS at 2 mM. Unlabelled shuttle PIP car- Ten μs of production simulation was performed for each of the three rier 2 (histone H1) was freshly reconstituted with water at 2 mM. PIP3 in starting conformations of the lipid bilayer. The final 1 μs of the trajec- a volume of 75 μl was mixed with 25 μl of PIP carrier and incubated at tory from each of the 3 setups was used to analyse the folded structure room temperature for 10 min. One hundred μl of PIP3–carrier complex of the CRAC1–basic patch–CRAC2 region of the IFITM3 model. We was added into Ifitm3+/+ or Ifitm3−/− B-ALL cells in a total volume of 5 ml clustered the conformations of IFITM3 by the root mean square devia- +/+ −/− and incubated for 24 h. PIP3-loaded Ifitm3 or Ifitm3 B-ALL cells were tion cut-off of 0.3 nm in the backbone coarse-grained particles in the subjected to colony-forming assay, as shown in Extended Data Fig. 8. coarse-grained simulations. We analysed the binding patterns of PIP2 and PIP3 in this most populated conformational cluster. We observed Lipid-binding assay that both PIP2 and PIP3 compete for the ‘basic patch’ of residues shown To assess the direct binding of IFITM3 to lipids, a lipid-binding assay in the amino acid sequence in Extended Data Fig. 9. We extracted three was performed using Membrane Lipid Strips (Echelon Biosciences) snapshots from this conformational cluster that represent the most according to the manufacturer’s instructions. In brief, membranes diverse patterns in PIP2 and PIP3 binding. were blocked in 5% fatty acid-free BSA (Sigma-Aldrich) in TBST (50 mM Tris-HCl,150 mM NaCl, and 0.1% Tween 20) for 1 h at room tempera- Details of the all-atom molecular dynamics simulations for ture in the dark followed by overnight incubation with 0.5 μg/ml of phospholipid binding recombinant proteins in blocking buffer at 4 °C with gentle agitation. To better understand the detailed binding conformation of PIP2 and After washing membranes 3 times for 30 min in TBST, membranes PIP3 to IFITM3 and to calculate their binding energies to IFITM3, we were incubated for 1 h with anti-GST tag polyclonal Ab (Thermo Fisher converted the three snapshots extracted from the coarse-grained Scientific) or anti-biotin monoclonal antibody (Cell Signaling Technol- simulations to an all-atom system using Martini tools51. The three ogy) antibodies, listed in Supplementary Table 10. Membranes were chosen snapshots were cut into 9 × 9-nm2 box centred at the IFITM3 incubated with alkaline-phosphatase-conjugated secondary antibodies unit from the coarse-grained simulations to preserve the local opti- (Invitrogen) and chemiluminescent substrate (Invitrogen) and were mized lipid environment. The lipid and protein were converted to an further detected by UVP BioSpectrum 810 Imaging System (Thermo all-atom model, and were resolvated into a 9 × 9 × 9-nm3 simulation Fisher Scientific). Recombinant GST-tagged protein was purchased box, neutralized by 0.15 M NaCl. Extended Data Figure 9 shows the from Sigma-Aldrich. Recombinant human GST–IFITM3 protein was detailed interaction between PIP2 or PIP3 with IFITM3 protein in an purchased from Abnova. Recombinant human IFITM3 fragments listed all-atom resolution base on these snapshots. The all-atom simula- in Supplementary Table 5 were synthesized at LifeTein. tions were performed using GROMACS package44 and CHARMM36 force field52, with the TIP3 water model. The nonbond interactions Preferential PIP3 accumulation and binding to IFITM3 using were calculated with a cut-off of 12 Å, and the particle-mesh Ewald multi-scale molecular dynamics simulations method51 was applied to solve long-range van der Waals interactions. The IFITM3 protein structure from residues 58 to 128 was modelled by Each system was gradually heated to 310 K with random initial velocities coarse-grained molecular dynamics simulation method in GROMACS, sampled from a Boltzmann distribution. The heating process lasted in explicit cell membrane mimic bilayer. There is no structure was avail- for 1ns, with temperature controlled by a Nosé–Hoover thermostat53, able for IFITM3 or for any close homologue. We calculated the hydro- followed by 30-ns equilibration in NPT ensemble with harmonic posi- phobicity for each position using the hydrophobic index of amino tion restraints on protein heavy atoms. The pressure was maintained at acids42 and averaged hydrophobicity over a sliding window of seven 1 bar in semi-isotropic environment, controlled by the Parrinello–Rah- amino acids. Previous structural studies on IFITM3 structure43 have man method. The restraint force was gradually reduced from 5 kcal/ shown there is only one transmembrane domain 2 that is inserted into mol to 0 kcal/mol with a −1 kcal/mol step per 5-ns window. The final the membrane. On the basis of this evidence, we used the topology frame of equilibration was taken for a production run. The produc- shown in Supplementary Fig. 9 as the starting structure. TM1 was gener- tion run was performed twice for 50 ns each, with 2 different random ated as an α-helix structure, and the CRAC motifs and linker region were initial velocities assigned to the equilibrated structure. An integration built as fully extended chain. The linker region was relaxed using 5,000 time step of 2 fs was used. The interaction energy between PIP3 with steps of coarse-grained simulation using GROMACS program44 with IFITM3 protein was calculated as the sum of electrostatic Coulombic Martini force field45 to remove stress in the system. We then added the energy and van der Waals potential energy averaged over the two 50-ns TM2 region as a helix and inserted TM2 into a cell-membrane-mimicking trajectories, totalling 100 ns. bilayer46. The composition and ratio of lipids for the mixed bilayer is POP C:DOPC:POPE:DOPE:CHOL:Sph:GM1 = 0.2:0.2:0.05:0.05:0.25:0.15:0.1 Interaction of PIP3 with the two basic residue patches for the outer leaflet of the bilayer, and 0.05:0.05:0.20:0.2:0.08:0.0 The PIP3-binding and AKT-signalling activation assays showed that 8:0.25:0.03:0.03:0.03 of POPC: DOPC: POPE: DOPE: POPS: DOPS: mutation of the K83/K104 patch to K83A/K104A had higher effect than CHOL: PIP1:PIP2:PIP3 for the inner leaflet. We generated three possible mutating the R85/R87/K88 patch to R85A/R87A/K88A. To further vali- starting setup for the mixed lipid bilayer with water in the upper and date the interaction strength difference between PIP3 and the two basic inner regions with CHARMM-GUI47,48. The charges in the system were patches, we converted all PIP1 and PIP2 in the simulation box to PIP3. neutralized with 0.15 M NaCl. We used nine replicates of the protein We further performed simulations on the IFITM3(K83A/K104A) and IFITM3 structure in the simulation box (Supplementary Fig. 9a). The on IFITM3(R85A/R87A/K88A) in the PIP3-only simulation box. Start- initial configuration was minimized using steepest decent method for ing from this structure, we performed equilibration of the system and 5,000 steps, and then equilibrated in NPT ensemble for 5 ns, at 303-K 5 all-atom molecular dynamics production simulations (each 200-ns temperature and 1 bar pressure. The temperature was controlled by long), using the protocol for all-atom molecular dynamics simulations Berendsen thermostat49 with coupling constant of 1 ps, and the pres- described in ‘Details of the all-atom molecular dynamics simulations sure was controlled by Berendsen barostat49 in semi-isotropic type for phospholipid binding’. We aggregated the final 100 ns of simula- with a coupling constant of 5 ps and a compressibility of 3 × 10−4 bar-1. tion trajectories from each of the 5 runs, which we added to 500 ns of Article molecular dynamics simulation trajectories for analysis. We calcu- within 10 min of taking cells out of the cell culture incubator. After a lated the interaction energy as the sum of electrostatic Coulombic and measurement of the untreated Jeko1 cells, a new patch of same cells −1 van der Waals potential energies between PIP3 and residues forming was obtained from the incubator, treated with 10 μg ml F(ab′)2 frag- the basic patch only (K83/K104 or R85/R87/K88). The interaction ener- ment goat anti-human μ chain (Jackson Immunoresearch) and the gies were averaged over the 500 ns of aggregated molecular dynamics cells were immediately (within about 1 min) loaded into the SMR for simulation trajectories for each system. We repeated these interac- measurement. Antibody-treated Jeko1 cells were measured for about tion energy calculations for the wild-type IFITM3 and for the mutant 7 min to ensure that the anti-human μ chain stimulated changes were K83A/K104A and R85A/R87A/K88A constructs. not reversed during the experiment. The SMR was briefly washed with PBS between each experiment. Wild-type Jeko1 cells were also meas- Contact-frequency heat-map calculations from molecular ured after fixation to obtain a positive control for cell stiffness. For dynamics simulations fixation, the cells were washed twice with PBS, mixed with 8% PFA for The important information that can be extracted from molecular 30 min, washed twice with PBS and stored at 4 °C before the acoustic dynamics simulations is the temporal frequency of PIP3 contacts with scattering measurements. the two basic residue patches. The persistence of these interactions has an important role in the accumulation of PIP3 by IFITM3. We cal- Quantification and statistical analysis culated the percentage of molecular dynamics snapshots that show Data are shown as mean ± s.d. unless stated. Statistical analysis was contacts between each of the residues in the basic patch. This is termed performed by GraphPad Prism 7 (GraphPad Software) using unpaired the contact frequency. We also calculated the percentage of molecu- two-tailed t test, or as indicated in figure legends. Significance was lar dynamics snapshots that show simultaneous contacts made by considered at P < 0.05. For in vivo transplantation experiments, the PIP3 with two or more residues in the two basic residue patches. We minimal number of mice in each group was calculated through use of generated a heat map using the frequencies calculated for the wild-type the ‘cpower’ function in the Hmisc package of R. Kaplan–Meier survival IFITM3 and in the alanine mutants. analysis was used to estimate overall survival with GraphPad Prism 7. Mantel–Cox log-rank test was used to compare the difference between Quantitative real-time PCR two groups. No mice were excluded. For patient overall survival analy- Bone marrow from a healthy donor was stained and sorted as previously sis, patients in each dataset were divided into two groups on the basis described54. Total RNA from cells was extracted using the RNA isolation of whether their expression was above or below the median level of kit from Macherey–Nagel. Complementary DNA was generated with the IFITM3 and Kaplan–Meier survival analysis was used to estimate overall qScript cDNA SuperMix (Quanta Biosciences). Quantitative real-time survival. The datasets used include patient-outcome data for B-ALL PCR was performed with FAST SYBR Green Master Mix (Applied Biosys- (Children’s Oncology Group (COG) P9906, n = 207; Eastern Coopera- tems) and the Vii7 real-time PCR system (Applied Biosystems) accord- tive Oncology Group (ECOG) E2993, n = 83; and St Jude, n = 15), mantle ing to standard PCR conditions. COX6B was used as a reference gene. cell lymphoma (Lymphoma/Leukemia Molecular Profiling Project (LLMPP), n = 92) and acute myeloid leukaemia (The Cancer Genome Acoustic scattering measurements Atlas (TCGA), n = 200). A log-rank test was used to compare survival Single-cell size-normalized acoustic scattering (SNACS) was meas- differences between patient groups. The R package ‘survival’ (version ured using a previously established microfluidic method, which has 2.35-8) was used for the survival analysis and Cox proportional hazards been shown to be specifically sensitive to cell-surface stiffness55. Full regression model in the R package for the multivariate analysis (https:// measurement details can be found in a previous publication55. In brief, www.r-project.org/). The investigators were not blinded to alloca- cells were flowed through a standing acoustic wave generated inside tion during experiments and outcome assessment. Experiments were a vibrating suspended microchannel resonator (SMR). The SMR is a repeated to ensure reproducibility of the observations. cantilever-based mass measurement tool56, which can also detect acous- tic scattering from cells when the cells transit through the acoustic Reporting summary wave. The cantilever vibration frequency was monitored, and its shift Further information on research design is available in the Nature was used to quantify the acoustic scattering from the cells as well as Research Reporting Summary linked to this paper. the buoyant mass of the cells. Before taking a set of measurements, the SMR was cleaned with 0.25% trypsin–EDTA for 20 min, followed by 5% bleach for 3-min and then a 5-min rinse with deionized H2O, to Data availability remove persistent biological debris. After cleaning, the SMR was pas- Patient-outcome data for B-ALL were obtained from the National sivated with 1 mg/ml PLL-g-PEG in H2O for 10 min at room temperature, Cancer Institute TARGET DATA Matrix of the COG Clinical Trial P9906 followed by a 5-min rinse with normal cell culture medium. During (GSE11877) (refs. 28,30), ECOG Clinical Trial E2993 (GSE5314) (ref. 57) and the measurement, all the samples were loaded into the SMR through St Jude Children’s Research Hospital (https://www.stjuderesearch. 0.005-inch-inner-diameter fluorinated ethylene propylene tubing. The org/site/data/ALL3/) (ref. 29). Patient-outcome data for mantle cell fluid flow across the SMR was driven by three independent electronic lymphoma were obtained from https://llmpp.nih.gov/MCL/ (ref. 58). pressure regulators and three solenoid valves. A consistent differen- Patient-outcome data for AML were obtained from TCGA Acute tial pressure was applied across the SMR to maintain constant shear Myeloid Leukaemia Project (http://www.cbioportal.org/study/ and data rate for cell measurement. The data displayed in Extended summary?id=laml_tcga_pub#clinical)59. Proteomics data have been Data Fig. 8a were obtained using a 350-μm-long cantilever with a deposited to the ProteomeXchange Consortium via the PRIDE part- 15 × 20-μm-sized channel inside of the cantilever and an approximately ner repository with the following accession numbers: cell-surface 200-ms transit time through the cantilever. All the regulators, valves proteome, PXD014691; phosphoproteome, PXD020696; and IFITM3 and data acquisition were controlled by custom software coded in interactomes, PXD020697. Levels of IFITM3 mRNA across human nor- LabVIEW 2017 (National Instruments) as detailed in a previous publica- mal and malignant B-lymphoid samples were obtained from http:// tion55. A parallel volume measurement using Coulter Counter (Beckman Amazonia.transcriptome.eu/. All other data are available from the Coulter) was carried out to quantify average cell volume, which was corresponding author upon reasonable request. Genome binding and used together with the single-cell buoyant mass measurements to cal- occupancy profiling from wild-type and IKDN stromal adherent pre-B culate SNACS for each cell, as previously reported55. All measurements cells were obtained from GSE86897. Immunohistochemistry images were carried out in normal cell culture medium at room temperature for IFITM3 levels in normal or malignant B cells were obtained from The Human Protein Atlas https://www.proteinatlas.org/. ChIP-seq data 52. Huang, J. & MacKerell, A. D. Jr. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J. Comput. Chem. 34, 2135–2145 (2013). of the genome-wide mapping of IKZF1 binding in patient-derived B-ALL 53. Martyna, G. J., Klein, M. L. & Tuckerman, M. Nosé–Hoover chains: the canonical ensemble xenograft cells were obtained from GSE58825. ChIP-seq data of the via continuous dynamics. J. Chem. Phys. 97, 2635–2643 (1992). genetic analysis of IKZF1 target genes and tumour suppressor function 54. Buchner, M. et al. Identification of FOXM1 as a therapeutic target in B-cell lineage acute + lymphoblastic leukaemia. Nat. Commun. 6, 6471 (2015). in Ph pre-B ALL cells were obtained from GSE90656. RNA-sequencing 55. Kang, J. H. et al. Noninvasive monitoring of single-cell mechanics by acoustic scattering. +/+ −/− G12D data for Ifitm3 and Ifitm3 BCR-ABL1 or NRAS B-ALL cells are avail- Nat. Methods 16, 263–269 (2019). able at GSE155305. RNA-sequencing data for Ptenfl/fl pre-B cells carrying 56. Burg, T. P. et al. Weighing of biomolecules, single cells and single nanoparticles in fluid. T2 T2 Nature 446, 1066–1069 (2007). 4-OHT-inducible Cre-ER or ER are available at GSE155618. Supple- 57. Juric, D. et al. Differential gene expression patterns and interaction networks in BCR-ABL- mentary Table 12 summarizes the accession numbers and publicly positive and -negative adult acute lymphoblastic leukemias. J. Clin. Oncol. 25, 1341–1349 deposited data from this study. All other data needed to evaluate the (2007). 58. Rosenwald, A. et al. The proliferation gene expression signature is a quantitative conclusions in the paper are available within the Article and its Sup- integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer plementary Information. Source data are provided with this paper. Cell 3, 185–197 (2003). 59. The Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of 27. Geng, H. et al. Integrative epigenomic analysis identifies biomarkers and therapeutic adult de novo acute myeloid leukemia. N. Engl. J. Med. 368, 2059–2074 (2013). targets in adult B-acute lymphoblastic leukemia. Cancer Discov. 2, 1004–1023 (2012). 28. Harvey, R. C. et al. Identification of novel cluster groups in pediatric high-risk B-precursor Acknowledgements We thank M. A. Lemmon for critical discussion and advice, and current acute lymphoblastic leukemia with gene expression profiling: correlation with genome- and former members of the Müschen laboratory for their support and helpful discussions. wide DNA copy number alterations, clinical characteristics, and outcome. Blood 116, Research in the Müschen laboratory is funded by the NIH/NCI through R35CA197628, 4874–4884 (2010). R01CA157644, R01CA213138 and P01CA233412, the Dr. Ralph and Marian Falk Medical 29. Ross, M. E. et al. Classification of pediatric acute lymphoblastic leukemia by gene Research Trust Transformational Awards Program through MCG-18447-19, the Leukemia and expression profiling. Blood 102, 2951–2959 (2003). Lymphoma Society through MCL-7000-18 and Blood Cancer Discoveries Grant 30. Kang, H. et al. Gene expression classifiers for relapse-free survival and minimal residual BCDG-20327-20, as well the Howard Hughes Medical Institute HHMI-55108547 (to M.M.). This disease improve risk classification and outcome prediction in pediatric B-precursor acute work was partly supported by NIH grants R35CA200422, R01CA251275, AI116585, AI116585S, lymphoblastic leukemia. Blood 115, 1394–1405 (2010). AI140718, AI140705, DE023926 and DE028521 (to J.U.J.), R01 AI104002 and R01 AI127513 (to 31. Borowitz, M. J. et al. Clinical significance of minimal residual disease in childhood acute M.S.D.), R01 GM117923 and R01 GM097261 (to N.V.), NCI R35 CA231958 (to D.M.W.), Wellcome lymphoblastic leukemia and its relationship to other prognostic factors: a Children’s Trust 110275/Z/15/Z (to T.P.M.) and the Paul G. Allen Frontiers Group and the Cancer Systems Oncology Group study. Blood 111, 5477–5485 (2008). Biology Consortium funding (U54-CA217377) from the National Cancer Institute (to S.R.M.). 32. Poddar, S., Hyde, J. L., Gorman, M. J., Farzan, M. & Diamond, M. S. 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Extended Data Fig. 1 | Ifitm3 expression is induced by oncogenic PI3K determined in the COG P9906 cohort, IFITM3 mRNA levels were compared in signalling and repressed by IKZF1. a, Changes of Ifitm3 mRNA levels were patients who were positive (n = 67) and negative (n = 124) for MRD. Two-tailed monitored in mouse splenic B cells upon BCR engagement (mean ± s.e.; n = 2). Wilcoxon. i–l, Patients with leukaemia and lymphoma from five clinical cohorts b, c, Ptenfl/fl pre-B ALL cells carrying 4-OHT-inducible Cre-ERT2 or ERT2 were were segregated into two groups based on higher (IFITM3high) or lower treated with 4-OHT and studied by RNA sequencing for Ifitm3 transcript (IFITM3low) than median IFITM3 mRNA levels. Overall survival was compared by expression (b) and western blot for protein levels of PTEN, AKT-pS473, AKT and two-tailed log-rank test. m, ChIP-seq enrichment of RNAPII and H3K4me3 at the IFITM3 (c; n = 3). d, STAT5 phosphorylated at Y694 (STAT5-pY694), STAT5 and Ifitm3 locus in pre-B cells (top) from Ikzf1exon5fl/fl mice upon Cre-mediated IFITM3 levels in patient-derived B-ALL cells (PDX2) measured by western deletion of Ikzf1 (GSE86897). Binding of IKZF1 to the promoter region of IFITM3 blotting upon imatinib treatment (n = 3). e, Scenario of the PI3K pathway as a was also analysed in ChIP-seq data from patient-derived B-ALL cells (bottom, positive regulator of IFITM3, which in turn amplifies BCR and oncogenic LAX2, GSE58825). n, Human B-ALL cells (BV173) carrying IKZF1 deletions were signalling. f, IFITM3 mRNA levels across human normal and malignant reconstituted with doxycycline-inducible IKZF1 or EV. Levels of IFITM3 were haematopoietic and B-lymphoid samples (source data and statistics are given assessed by western blotting upon doxycycline-induction (n = 3). o, in Supplementary Table 2). Two-tailed t-test. g, IFITM3 mRNA levels in pre-B Multivariate analysis of established risk factors in children with B-ALL (COG cells from healthy donors and samples from patients with B-ALL were P9906, n = 207) including mutation or deletion of IKZF1. Patients (n = 207) were compared for two clinical cohorts (ECOG E2993 and St Jude). In ECOG E2993, separated into IKZF+/+ or IKZF-deleted groups, then further segregated based bone marrow samples were obtained at diagnosis before treatment from 83 on higher or lower than median expression levels of IFITM3. The comparison of adults with B-ALL with a confirmed purity of >90% leukaemic blasts. For the St these four groups established IFITM3 mRNA levels as an independent risk Jude datasets, 15 samples from children with B-ALL before treatment were factor regardless of IKZF1-deletion status (two-sided log-rank test; P = 0.0045). compared to flow-sorted pre-B cells from bone marrow aspirates of healthy c, d, n, For gel source data, see Supplementary Fig. 1. donors. Two-tailed Wilcoxon. h, Minimal residual disease (MRD) was Extended Data Fig. 2 | Ifitm3 is essential for the development of B1 and cell-permeant Fluo-4 dye; MFI compared between replicates (left; n = 3). germinal centre B cells. a, Hardy fractions of B cell subsets isolated from bone Surface expression of CD19 following deletion of IFITM3 in Jeko1 MCL cells, marrow of Ifitm3+/+ and Ifitm3−/− littermates analysed by flow cytometry (n = 3). MFIs for CD19 indicated (right; n = 3). e, Jeko1 MCL cells were electroporated b, Surface expression of IgM, CD20, CD19, IgD, CD2 and CD21 measured by flow with non-targeting RNP (Cas9-gRNA ribonucleoproteins, gNT) or cytometry in enriched bone marrow (GR1−, Nk1.1− and B220+) and splenic B cells IFITM3-targeting RNP complex (gIFITM3). Following electroporation, (CD3− and B220+) from Ifitm3+/+ or Ifitm3−/− mice (n = 7; mean ± s.d.). Mean MCL cells were treated with vehicle (DMSO) or 25 nmol l−1 of dasatinib for 3 h. −1 fluorescence intensities (MFI) values for individual measurement compared by Cells were stimulated with 10 μg ml of anti-human IgM F(ab′)2 for the indicated two-tailed t-test. c, Ca2+ mobilization from cytoplasmic stores in response to time points and subjected to co-immunoprecipitation with an anti-CD19 BCR (IgM) engagement was measured in Ifitm3+/+ and Ifitm3−/− splenic B cells. antibody. Immunoblots were performed to measure levels of CD19 tyrosine Ca2+ release was induced by addition of 10 μg ml−1 anti-mouse IgM 60 s after phosphorylation and binding of LYN to CD19. Levels of IFITM3, SRC-pY416 and acquisition of background fluorescence. Ca2+ release was measured over 300 s LYN were assessed in whole-cell lysates (10% input) with β-actin as loading with cell-permeant Rhod-2 dye; MFI compared between replicates (n = 3). d, control. (n = 3; gel source data in Supplementary Fig. 1). f–h, Relative fractions Ca2+ mobilization in response to BCR engagement measured upon CRISPR– (left) and absolute cell counts (right) of total B1 (f) and B1a (g) cells in the Cas9-mediated deletion of IFITM3 in Jeko1 MCL cells. Ca2+ release upon addition peritoneal cavity and marginal-zone B cells (h) in spleen of Ifitm3+/+ and Ifitm3−/− −1 of 10 μg ml of polyclonal F(ab′)2 anti-human IgM was measured for 300 s with littermates (n = 5) are shown (means ± s.d.; two-tailed t-test). Article

Extended Data Fig. 3 | Ifitm3-deficient B-ALL cells exhibit an anergic Statistical significance was determined by two-tailed Kolmogorov–Smirnov phenotype and compensatory upregulation of Ifitm1 and PI3K signalling test. d, Patient-derived B-ALL cells (PDX2) were transduced with N-terminally molecules. a, Numbers of viable Ifitm3+/+ and Ifitm3−/− BCR-ABL1 or NRASG12D Flag-tagged or C-terminally HA-tagged IFITM3 constructs. Combinations of B-ALL cells were counted by Trypan blue dye exclusion (n = 3; mean ± s.d.; intracellular and surface staining were performed to examine IFITM3 topology two-tailed t-test). b, RNA sequencing was performed for Ifitm3+/+ and Ifitm3−/− at the cell membrane. e, Patient-derived B-ALL cells (PDX2) were transduced BCR-ABL1 and NRASG12D B-ALL cells. Relative rlog-normalized gene expression with C-terminal HA-tagged IFITM3 or the IFITM3(Y20E) phosphomimetic. values for all strongly differentially expressed genes (P < 1 × 10−5 and Combinations of intracellular and surface staining, with or without SRC-kinase log2-transformed fold change >1; Wald test with Benjamini–Hochberg inhibition by dasatinib, were performed to examine IFITM3 topology at the cell correction) in both BCR-ABL1 and NRASG12D conditions plotted as a heat map membrane and its regulation by SRC kinases. d, e, Representative plots from with row scaling. B-cell-signalling-related genes are labelled in red, three independent experiments. f, A scenario of the topology of IFITM3 anergy-related genes are labelled in blue and PI3K-signalling-related genes are regulated by SRC (LYN) or oncogenic tyrosine kinases (BCR–ABL1) at the labelled in grey. c, Gene set enrichment analysis for genes ranked by ratio of plasma membrane is shown. Phosphorylation of Y20 hinders the recognition of −/− +/+ 20 23 Ifitm3 to Ifitm3 as log2-transformed fold change; red lines indicate running YEM endocytosis motif by the AP-2 complex, thereby antagonizing enrichment score (right axis), grey bars indicate fold change (left axis). endocytosis and endosomal trafficking of IFITM3. Extended Data Fig. 4 | See next page for caption. Article Extended Data Fig. 4 | IFITM3 amplifies PI3K signalling downstream of BCR acquisition of background fluorescence. Surface expression of CD3 was and integrin receptors. a, Volcano plot of differentially phosphorylated measured following deletion of IFITM3 in Jurkat cells (right). MFIs for CD3 are proteins in patient-derived B-ALL (PDX2) cells transduced with IFITM3(Y20E) indicated. Representative plots from three independent biological compared to empty vector (EV) control (n = 3; Wald test with Benjamini– experiments are shown. MFI values for individual measurement were Hochberg correction). b, F(ab) fragments of the anti-HA antibody or isotype compared by two-tailed t-test. d, Surface proteins on Ifitm3+/+ and Ifitm3−/− control were purified and their identity confirmed by western blot (left). Eight B-ALL cells were labelled with biotin and enriched with streptavidin affinity million patient-derived B-ALL (PDX2) cells carrying IFITM3–HA, IFITM3(Y20E)– pull-down followed by on-bead trypsin digestion, mass spectrometry and HA or EV control were resuspended in complete medium and treated with quantified with label-free quantification. Differentially expressed cell surface either 2.5 μg ml−1 of full antibodies, F(ab) fragments of anti-HA or isotype proteins on Ifitm3+/+ and Ifitm3−/− B-ALL cells are shown with the mean control for the indicated times. Levels of AKT-pS473, AKT and HA-tagged IFITM3 difference of label-free quantification plotted against the P value (Welch’s t- were assessed by western blots using β-actin as loading control (right). Data test). All experiments were performed in biological triplicates. e–g, Validation from three independent experiments. For gel source data, see Supplementary of differential expression of surface receptors between Ifitm3+/+, Ifitm3−/− and 2+ Y20E Fig. 1. c, Ca mobilization in response to TCR engagement using CD3ɛ-specific Ifitm3 -overexpressing B-ALL cells. Flow cytometry analyses show surface antibodies was measured upon CRISPR–Cas9-mediated deletion of IFITM3 in expression of BCR signalling components (e), integrins and adhesion receptors T-ALL cells (Jurkat) (left). Ca2+ release from cytoplasmic stores was induced by (f) and other surface receptors (g) in Ifitm3+/+, Ifitm3−/− and B-ALL cells −1 adding 10 μg ml of monoclonal (OKT3) anti-human CD3ɛ at 50 s after expressing IFITM3(Y20E) (n = 3). Extended Data Fig. 5 | Inducible membrane translocation of CD19 does not were selected. Cd19−/− B-ALL cells were transduced with CD19–ERT2, a fusion of the rescue defective SRC and PI3K signalling in Ifitm3-deficient B cells. a, Surface ER-ligand binding domain to the C terminus of CD19, or ERT2 as empty vector expression of CD19 was assessed by flow cytometry following forced expression control. Reconstitution of Cd19−/− B-ALL cells with CD19–ERT2 resulted in stable of CD19 for >1 week in Ifitm3+/+ and Ifitm3−/− B-ALL cells. b, Western blot analyses expression of the fusion proteins that were retained in complex with cytoplasmic of AKT-pS473, AKT, MYC and BCL2 upon forced expression of CD19 for >1 week in heatshock proteins. Addition of 4-OHT released CD19–ERT2 from its cytoplasmic mouse Ifitm3+/+ and Ifitm3−/− B-ALL cells. c, d, Colony-forming ability (c) and heatshock chaperone and enable cell surface expression within 30 min of 4-OHT cell cycle progression (d) of Ifitm3+/+ and Ifitm3−/− B-ALL cells upon forced addition. g, To test the effect of inducible CD19 membrane translocation in expression of CD19 for >1 week was examined. c, Colony numbers for individual Ifitm3−/− B-ALL cells, Ifitm3+/+ and Ifitm3−/− B-ALL cells were transduced with CD19– measurement were compared by two-tailed t-test. d, Numbers indicate ERT2 or ERT2 empty vector control. 4-OHT-mediated translocation of CD19 to the percentage of cells in S phase. e, Numbers of viable Ifitm3+/+ and Ifitm3−/− B-ALL cell surface was assessed by flow cytometry for the indicated times (0 to 3 h). h, cells following forced expression of CD19 were counted using the Trypan blue Ifitm3+/+ and Ifitm3−/− B-ALL cells were transduced with CD19–ERT2 or ERT2 empty dye exclusion method (left). Ifitm3−/− B-ALL cells were transduced with vector control. Cells were treated for 0, 1 and 3 h with 4-OHT for GFP-tagged constructs for expression of CD19 with an intact (Y531) or mutant surface-translocation of CD19. Cell lysates from these populations were analysed (Y531F) PI3K-activation motif in its cytoplasmic tail. Relative changes by western blot for CD19-pY531, CD19, SRC-pY416, LYN, AKT-pS473, AKT, MYC and of GFP+ cells (transduced with CD19 or CD19(Y531F)) were plotted over time BCL2. While CD19–ERT2 reconstitutes CD19 protein levels in Ifitm3−/− B-ALL cells (means ± s.d.). a–e, Data from three independent experiments. b, For gel source and restores rapid translocation to the cell surface (g), this change alone was not data, see Supplementary Fig. 1. f, Mouse Cd19−/− B-ALL clones were generated by sufficient to induce proper phosphorylation of CD19, SRC kinases and PI3K electroporation of mouse B-ALL cells with CD19-targeting RNP (Cas9-gRNA signalling via AKT. f–h, Data from three independent experiments. h, For gel ribonucleoproteins, gCd19 ALL) and single-cell clones with biallelic deletion source data, see Supplementary Fig. 1. Article

Extended Data Fig. 6 | IFITM3 links components of the BCR and integrin integrin (blue) and PI3K signalling (grey) are highlighted. Data from three receptor pathways to PI3K signalling. a, Schematic of HA-tagged-IFITM3(Y20E)– independent biological replicates. e, PLAs were performed with Jeko1 MCL cells BirA fusion proteins used for TurboID interactome analyses. BirA (biotin ligase) upon engagement of BCR. Jeko1 MCL cells were stimulated by BCR engagement for was fused to N-terminal IFITM3 carrying the phosphomimetic Y20E mutation for 0, 3 and 30 min, then fixed, permeabilized and assessed for the proximity of membrane localization. b–d, HA–IFITM3(Y20E)–BirA or HA–BirA controls were CD79B to IFITM3. Representative microscopic images with PLA signal (red dot) expressed in PDX2 B-ALL (b) or Jeko1 MCL cells (c, d). c, d, Cells were incubated with and nuclei stained with DAPI as blue are shown. LAMP1 was used as a marker for exogenous biotin for 10 min upon IFITM3 (anti-HA) or BCR (anti-IgM) engagement. endosomes to distinguish plasma-membrane-bound from endosomal localization IFITM3(Y20E) interactome analyses identified interacting proteins by mass of CD79B:IFITM3 complexes. Scale bars, 5 mm. Data from three independent spectrometry, plotted based on significance and log2-transformed fold replicates. enrichment over EV control. Essential interactors as BCR component (red), Extended Data Fig. 7 | See next page for caption. Article Extended Data Fig. 7 | Ifitm3 functions as a central effector of B cell thousand Ifitm3+/+ or Ifitm3−/− B-ALL cells carrying IK6 or EV were cultured on adhesion. a, Homotypic aggregation was studied in Ifitm3−/− B-ALL cells that OP9 stroma cells. Ratios of adherent cells to nonadherent cells were calculated. were reconstituted with C-terminal HA-tagged IFITM3, IFITM3(Y20E) or empty Data from three independent biological experiments (right) were analysed and vector (EV) and incubated with anti-CD19, anti-HA antibodies or isotype assessed by two-tailed t-test (means ± s.d.). d, Representative images of control for 24 h. Data from three independent experiments. b, Ifitm3+/+ or adherent B-ALL cells on OP9 stroma are shown. Round and light-refracting cells Ifitm3−/− B-ALL cells were transduced with GFP-tagged IK6 or GFP alone. IK6 are adherent B-ALL cells attached to stroma cells. Dark and round cells are levels in flow-sorted GFP+ cells assessed by western blot analysis using β-actin adherent B-ALL cells incorporated into stromal layer. Data from three as loading control (right). Ten thousand Ifitm3+/+ or Ifitm3−/− B-ALL cells carrying independent biological replicates. e, Surface expression levels of integrins on IK6 or EV were plated for colony forming assays. Colonies were imaged and adherent B-ALL cells were measured by flow cytometry. MFI values are counted after 7 days. Representative images are shown with colony numbers. indicated for individual measurements. Data from three independent Data from three independent experiments and assessed by two-tailed t-test biological replicates. (means ± s.d.). For gel source data, see Supplementary Fig. 1. c, One hundred Extended Data Fig. 8 | IFITM3 functions as a PIP3 scaffold and mediated paraformaldehyde (PFA) was used as a positive control. Representative plots cell-membrane stiffening upon BCR engagement. a, SNACS was measured from three independent experiments are shown (median levels in red dotted using a previously established microfluidic method as a metric for cell surface line). Statistical significance was determined by two-tailed t-test. Numbers stiffness. Jeko1 mantle cell lymphoma cells were flown through a standing indicated cells studied for SNACS measurement. b, Ifitm3+/+ and Ifitm3−/− B-ALL acoustic wave generated inside a vibrating suspended microchannel resonator. cells were incubated with 30 μM of the PIP3 carrier histone H1 or PIP3–histone The cantilever vibration frequency was monitored, and its shift was used to H1 complex for 30 min. Levels of CD19-pY531, CD19, SRC-pY416, LYN, AKT-pS473, quantify the acoustic scattering from the cells as well as the buoyant mass of AKT and MYC were measured by western blot using β-actin as loading control. the cells. The data displayed was obtained using a 350-μm-long cantilever with Data from three independent replicates. For gel source data, see 15 × 20-μm-sized channel inside of the cantilever and an approximately 200-ms Supplementary Fig. 1. c, Colony-formation assays were performed for Ifitm3+/+ transit time through the cantilever. All the regulators, valves and data and Ifitm3−/− B-ALL cells that were treated with 30 μM of PIP3–histone H1 or the acquisition were controlled by custom software coded in LabVIEW 2017 shuttle protein histone H1 (H1) alone. Photomicrographs and colony numbers (National Instruments). A parallel volume measurement using Coulter Counter per 10,000 plated cells are shown. Data are presented as means ± standard was carried out to quantify average cell volume, which was used together with derivation (s.d.) from three independent experiments. Statistical significance the single-cell buoyant mass measurements to calculate SNACS for each cell. was determined by two-tailed t-test. d, Flow cytometry analyses of surface Jeko1 mantle cell lymphoma cells without (IFITM3+/+) and with (IFITM3−/−) expression of CD19, CD44, CD25 and CD44 on Ifitm3+/+ and Ifitm3−/− B-ALL cells CRISPR–Cas9-mediated deletion of IFITM3 were kept under cell culture treated with 30 μM of PIP3–histone H1 or the shuttle protein histone H1 (H1) conditions and treated with an anti-IgM antibody. Fixation with alone for 72 h. Data from three independent replicates. Article

Extended Data Fig. 9 | Modelling of binding of IFITM3 to PIP2 and PIP3. batches measured in all-atom molecular dynamics simulations is indicated a–c, The hierarchical scheme of molecular dynamics simulations to delineate (Supplementary Table 6). d, e, The average interaction energy of PIP2 (green) the structural and dynamical basis of PIP2 and PIP3 binding to IFITM3 (yellow). or PIP3 (red) with IFITM3 (residues 57–128) was measured in all-atom molecular a, Coarse grained simulations of IFITM3 in composite cell membrane. The side dynamics simulations. The one-residue contact indicates the conformation of views of the lipid bilayer are shown. The lipid raft markers are shown in colour PIP2 or PIP3 binding with only one basic residue in the basic amino patch with GM1 (pink), cholesterol (green) and sphingomyelin (blue). The grey KSRDRK of IFITM3. The two-residue contact refers to the binding surface represents all the other lipids. b, The simulation cell extracted from the conformation of PIP2 or PIP3 that show contacts with two basic residues. coarse grained simulation as the starting structure for all-atom molecular Representative plots from at least four independent experiments are shown dynamics simulations. The IFITM3 protein is shown in yellow and the PIP2 and (mean ± s.d.). In d, P values were determined by two-tailed t-test. Populations PIP3 are shown in green and red stick representations. c, Close up view of one of showing one- or two-residue contacts of PIP2 (green) or PIP3 (red) to IFITM3 the predicted binding poses of PIP2 and PIP3 in the most populated were quantitated from all-atom molecular dynamics simulations. The conformation of IFITM3. The dashed lines shown are the PIP2 or PIP3 contacts population density was assessed by the normalization of number of events with the basic residues in IFITM3 (top). The amino acid sequence of IFITM3 in with the total number of frames. Representative plots from three independent the stretch of residues between TM1 and TM2 from 57 to 128 modelled in this experiments are shown (mean ± s.d.). In e, statistical significance was work is shown. The CIL region is boxed and basic amino acids are highlighted determined by two-tailed t-test. with blue. The average interaction energy of PIP2 or PIP3 with the two basic Extended Data Fig. 10 | IFITM3-mediated PI3K signalling downstream of increased in IFITM3(Y20E) over EV. The analysis was repeated for the K83A BCR and integrin receptors depends on K83–K104 but not on R85, R87 (red) and R85A (green) mutants of IFITM3. The light grey line indicates and K88 residues. a–c, Levels of differentially phosphorylated proteins in background variance observed between EV replicates. Shifts that were caused patient-derived B-ALL (PDX2) cells transduced with IFITM3(Y20E), by the K83A and R85A mutants are indicated by arrows. Overall changes in IFITM3(Y20E/K83A), IFITM3(Y20E/R85A) or empty vector (EV) control were distribution (shifts) between IFITM3(Y20E) and IFITM3(Y20E/K83A) and identified by mass spectrometry (n = 3). Relative abundance values are plotted IFITM3(Y20E/R85A) mutants were measured by two-tailed Kolmogorov– for all sites ranked by fold change as indicated; phosphosites of interest are Smirnov test. e, Schematic of BirA (engineered biotin ligase) was fused at highlighted. b, FSEA ranked by log2-transformed fold change are shown for N-terminal ends of HA-tagged IFITM3(Y20E) or its K83A mutant and expressed phosphosites in PI3K signalling (grey), BCR signalling (red) and integrin and in PDX2 B-ALL or Jeko1 MCL cells. e–g, Interactomes of IFITM3(Y20E) and its adhesion receptor elements (blue). Statistical significance was determined by K83A mutant were compared in PDX2 B-ALL cells upon IFITM3 engagement two-tailed Kolmogorov–Smirnov test. d, Cumulative distribution frequencies (anti-HA) (e) or in Jeko1 MCL cells upon IFITM3 (anti-HA) (f) or BCR (anti-IgM) for log2-transformed fold changes in phosphosite abundance between (g) engagement. Phosphosites of interest, including BCR signalling, PI3K IFITM3-transduced and EV conditions were calculated for all sites and globally signalling and integrins and adhesion receptor elements are highlighted.      

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