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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2782-2782
Author(s):  
Jaewoong Lee ◽  
Gang Xiao ◽  
Kadriye Nehir Nehir Cosgun ◽  
Huimin Geng ◽  
Ning Ma ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Membrane ◽  
B Cell ◽  
Src Kinase ◽  
Surface Expression ◽  
Pi3k Signaling ◽  
Mrna Levels ◽  
Poor Clinical Outcome ◽  
Advisory Committees ◽  
Equity Ownership

Background: The interferon-inducible transmembrane protein Ifitm3 (also known as Fragilis) plays an important role in primordial germ cell specification and functions as critical antiviral effector preventing fusion of virion-containing vesicles with endosomal membranes. Results: Here, we identified Ifitm3 as a biomarker of poor clinical outcome in patients with various B-cell malignancies. We found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.011; n=207) and adults (ECOG E2993; P=0.017; n=55) with B-ALL. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B-ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of Ifitm3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In mechanistic studies, we identified N-terminal phosphorylation at endocytic motif (20YEML23) by Src-kinases induced Ifitm3 cell surface accumulation during normal activation or oncogenic transformation of B-cells. In a lipid-binding assay in vitro, recombinant IFITM3 directly bound to PI(3,4,5)P3 but not any other phospholipids. In the cell membrane, therefore, IFITM3 functioned as a scaffold for PI(3,4,5)P3 and was essential for Src-kinase and PI3K signaling, as well as lipid raft formation and surface expression of multiple raft-associated receptors. Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of Src-kinase signaling. In the absence of Ifitm3, resting B-cell populations developed normally. However, consistent with defective Src-kinase and PI3K signaling, Ifitm3-/- B-cells failed to form germinal centers and to give rise to antigen-specific humoral immune responses. Likewise, Ifitm3-/- B-cell precursors were resistant to malignant transformation and lacked the ability to initiate BCR-ABL1- and NRASG12D-driven leukemia. Conversely, an Ifitm3-phosphomimetic of Src-kinase phosphorylation induced constitutive cell membrane localization, triggered oncogenic Src-PI3K signaling and initiated overt leukemia in pre-malignant B-cells. Conclusions: We conclude that Src-kinase-mediated phosphorylation of Ifitm3 induces a dynamic switch from antiviral effector functions in endosomes to lipid raft signaling at the cell membrane. While membrane-bound Ifitm3 is critical for normal B-cell receptor signaling and antigen-specific B-cell responses, its role as signal amplifier can be leveraged by multiple oncogenes for malignant B-cell transformation. Figure Disclosures Nix: UCSF: Patents & Royalties. Chen:Genovel Biotech Corp: Other: scientific founder and Chairman. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

IFITM3 Is a Central Regulator of Lipid Raft Signaling and Essential for CD19 Surface Expression and PI3K Signaling in Human B Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2738-2738
Author(s):  
Jae-Woong Lee ◽  
Huimin Geng ◽  
Kadriye Nehir Cosgun ◽  
Lai N Chan ◽  
Zhengshan Chen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
B Cell ◽  
Lipid Rafts ◽  
Transmembrane Protein ◽  
Cell Lineage ◽  
Surface Expression ◽  
Pi3k Signaling ◽  
Mrna Levels

Abstract Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) was identified as interferon-inducible molecule in the context of viral infection. Endosomal membrane localized IFITM3 appear to prevent fusion events of intraluminal viral particles to the endosomal membrane through accumulation of cholesterol which makes membrane more rigid. We recently found that IFITM3 is a dual-pass transmembrane protein expressed on B cell lineage ALL cells. Thereby IFITM3 is associated with known B cell co-receptors including CD19, CD81 and CD21. While the significance of this association was unknown, we found that IFITM3 is required for surface expression of the B cell antigen CD19. Although immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013), in some cases, CD19-specific engineered chimeric antigen receptors (CART19) treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Results: Studying IFITM3 mRNA levels in B cell lineage ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of ALL relapse and positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRAS. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K signaling in both normal and malignant B cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In vivo transplant setting, Ifitm3-/- pre-B ALL cells failed to initiate fetal leukemia in transplant-recipient mice. In mechanistic study, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT. Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe caused accumulation of IFITM3 at plasma membrane and led to constitutive activation of CD19/PI3K-AKT signaling. In addition to the gain-of-function mutants, extracellularly exposed C terminus was further stimulated by agonistic antibodies against IFITM3, which triggers CD19/PI3K-AKT signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells. Through Filipin based cholesterol staining, we found Ifitm3-/- pre-B cells have low levels of cholesterol at plasma membrane, which causes disruption of lipid rafts formation with decreased levels of ganglioside GM1. Thereby, Inadequate CD19/BCR co-receptor signaling caused by disruption of lipid rafts homeostasis by Ifitm3 deficiency results in critical developmental defects of peritoneal B1 cell compartment in vivo. Conclusion: These findings identify novel role of the IFITM3 surface receptor maintaining lipid rafts and CD19 surface expression. IFITM3-dependent lipid raft stability and CD19 surface expression were essential for normal and oncogenic PI3K signaling. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K in normal pre-B cells and various subtypes of human pre-B ALL. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 552-552
Author(s):  
Jae-Woong Lee ◽  
Huimin Geng ◽  
Derek S Dinson ◽  
Gang Xiao ◽  
Kadriye Nehir Cosgun ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Membrane ◽  
B Cell ◽  
Lipid Rafts ◽  
Transmembrane Protein ◽  
Cell Receptor ◽  
Mrna Levels ◽  
Viral Immunity ◽  
Oncogenic Signaling ◽  
Ras Pathway

Abstract Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts. Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to "trap" intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion. While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In mechanistic studies, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization. Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. Figure Figure. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

scholarly journals First-in-Human, Phase 1/2 Trial to Assess the Safety and Clinical Activity of Subcutaneous GEN3013 (DuoBody®-CD3×CD20) in B-Cell Non-Hodgkin Lymphomas

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 758-758 ◽  
Author(s):  
Pieternella Lugtenburg ◽  
Rogier Mous ◽  
Michael Roost Clausen ◽  
Martine E.D. Chamuleau ◽  
Peter Johnson ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Research Funding ◽  
Clinical Activity ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Introduction: CD20-specific monoclonal antibodies (mAbs) have demonstrated efficacy in the treatment of B-cell non-Hodgkin lymphomas (B-NHL); however, a significant proportion of patients (pts) present with refractory disease or will experience relapse. GEN3013 (DuoBody®-CD3×CD20) is the first subcutaneously administered IgG1 bispecific antibody (bsAb) that targets the T-cell surface antigen CD3 and the B-cell surface antigen CD20, triggering T-cell-mediated killing of B cells. In vitro, GEN3013 efficiently activates and induces cytotoxic activity of CD4+ and CD8+ T cells in the presence of B cells (Hiemstra et al. Blood 2018), and results in long-lasting depletion of B cells in cynomolgus monkeys. Subcutaneous (SC) GEN3013 in cynomolgus monkeys resulted in lower plasma cytokine levels, and similar bioavailability and B-cell depletion, compared with intravenous administration. GEN3013 has higher potency in vitro than most other CD3×CD20 bsAbs in clinical development (Hiemstra et al. HemaSphere 2019). SC GEN3013 in pts with B-NHL is being evaluated in a first-in-human, Phase 1/2 trial (NCT03625037), which comprises a dose-escalation part and a dose-expansion part. Here we report preliminary dose-escalation data. Methods: Pts with CD20+ B-NHL with relapsed, progressive, or refractory disease following anti-CD20 mAb treatment, and ECOG PS 0-2 were included. During dose escalation, pts received SC GEN3013 flat dose in 28-day cycles (q1w: cycle 1-2; q2w: cycle 3-6; q4w thereafter) until disease progression or unacceptable toxicity. Risk of cytokine release syndrome (CRS) was mitigated with the use of a priming dose and premedication with corticosteroids, antihistamines, and antipyretics. Primary endpoints were adverse events (AEs) and dose-limiting toxicities (DLTs). Secondary endpoints included pharmacokinetics (PK), immunogenicity (anti-drug antibodies [ADA]), pharmacodynamics (PD) (cytokine measures; laboratory parameters), and anti-tumor activity (tumor size reduction; objective and best response). Results: At data cut-off (June 28, 2019), 18 pts were enrolled into the dose-escalation part of the trial, with safety data available for pts receiving doses starting at 4 µg. Most pts had diffuse large B-cell lymphoma (DLBCL; n=14) and were heavily pre-treated; 10 pts had received ≥3 prior lines of therapy (overall median [range]: 3 [1-11]). The median age was 58.5 years (range: 21-80), and 13 pts were male. At a median follow-up of 1.9 months, pts received a median of 5 doses (range: 1-14); treatment is ongoing in 6 pts. Twelve pts discontinued treatment due to progressive disease. Six pts died (2 during treatment, 4 during survival follow-up), all due to disease progression and unrelated to treatment. The most common (n≥5) treatment-emergent AEs were pyrexia (n=8), local injection-site reactions (n=7), diarrhea (n=5), fatigue (n=5), and increased aspartate aminotransferase (n=5). The most common Grade (G) 3/4 AEs were anemia (n=3) and neutropenia (n=3). Despite increasing GEN3013 doses, all CRS events were non-severe (initial observation: 3/8 pts, G1: n=1, G2: n=2; following modification of premedication plan [corticosteroids for 3 days]: 6/10 pts, G1: n=4, G2: n=2). Increases in peripheral cytokine (IL6, IL8, IL10, IFNγ, TNFα) concentrations after GEN3013 dosing correlated with clinical symptoms of CRS in most pts. No pts had tumor lysis syndrome or neurological symptoms. No DLTs were observed. GEN3013 PK profiles reflect SC dosing; Cmax occurred 2-4 days after dosing. No ADAs were detected. PD effects following GEN3013 dosing were observed at dose levels as low as 40 µg and included rapid, complete depletion of circulating B cells (if present after prior anti-CD20 therapy) and peripheral T-cell activation and expansion. The first evidence of clinical activity was observed at a dose level of 120 µg, with complete metabolic response observed in a pt with DLBCL. Conclusions: Subcutaneously administered GEN3013, a potent CD3×CD20 bsAb, shows good tolerability and early evidence of clinical activity at low dose levels in heavily pretreated pts with relapsed or refractory B-NHL. All CRS events were non-severe and did not lead to discontinuation. No DLTs were observed. Dose escalation is ongoing; updated data will be presented. Dose expansion will begin upon determining the recommended Phase 2 dose (RP2D) (NCT03625037). Disclosures Lugtenburg: Janssen Cilag: Honoraria; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Genmab: Consultancy, Honoraria; BMS: Consultancy; Takeda: Consultancy, Honoraria, Research Funding. Mous:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sandoz: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Takeda: Honoraria, Research Funding; Janssen Cilag: Consultancy, Honoraria; MSD: Honoraria; Gilead: Consultancy, Honoraria, Research Funding. Clausen:Abbvie: Other: Travel grant to attend ASH 2019. Johnson:Boehringer Ingelheim: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Epizyme: Honoraria, Research Funding; Incyte: Honoraria; Takeda: Honoraria; Genmab: Honoraria; Bristol-Myers Squibb: Honoraria; Kite: Honoraria; Novartis: Honoraria. Rule:Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Astra-Zeneca: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Napp: Consultancy; Kite: Consultancy. Oliveri:Genmab: Employment, Equity Ownership. DeMarco:Genmab: Employment, Equity Ownership. Hiemstra:Genmab: Employment, Equity Ownership, Other: Warrants. Chen:Genmab: Employment. Azaryan:Genmab: Employment. Gupta:Genmab: Employment, Equity Ownership. Ahmadi:Genmab Inc: Employment, Other: stock and/or warrants. Hutchings:Incyte: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Research Funding; Pfizer: Research Funding.

scholarly journals Efficacy and Safety Results of a Phase 1 Study of 177 lu-DOTA-HH1 (Betalutin®) with and without HH1 Pre-Dosing for Patients with Relapsed CD37+ Non-Hodgkin B Cell Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5118-5118
Author(s):  
Arne Kolstad ◽  
Ulf Madsbu ◽  
Bjørg Bolstad ◽  
Caroline Stokke ◽  
Tore Bach-Gansmo ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Lymphoma ◽  
Safety Data ◽  
Efficacy And Safety ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Equity Ownership

Abstract Background: CD37 is an internalizing transmembrane antigen highly expressed by normal B cells and on most of B-cell malignancies, and represents an interesting therapeutic target for the treatment of B-cell NHL. 177Lu-DOTA-HH1 (Betalutin®) is a novel CD37-targeting antibody radionuclide conjugate in clinical development. It consists of a CD37-binding murine IgG1 antibody HH1 labelled with the short-ranged beta-emitter lutetium-177 (T½ = 6.7 days) chelated to DOTA. 177Lu-DOTA-HH1 is delivered in a ready-to-use formulation. Efficacy and safety data of patients (pts) receiving 177Lu-DOTA-HH1 with HH1 pre-dosing, as well as new efficacy and safety data from pts receiving 177Lu-DOTA-HH1 without HH1 pre-dosing will be presented. Methods: Pts with relapsed incurable CD37 positive NHL of follicular grade I-IIIA, marginal zone, mantle cell, lymphoplasmacytic and small lymphocytic subtypes and with platelet counts ≥ 150 x109/l were eligible for inclusion in the study. In a 3+3 study design pts received rituximab (375 mg/m2) day 1 and 8 in order to deplete normal B cells. On day 29 pre-dosing with HH1 (50 mg, cold CD37 antibody) was administered before 177Lu-DOTA-HH1 injection (Arm 1). In Arm 2 177Lu-DOTA-HH1 was administered without HH1 pre-dosing on day 29. The starting doses for Arm 1 and 2 were 10 MBq/kg b.w. and 15 MBq/kg b.w, respectively. Pts enrolment has been completed (n=13) in Arm 1 with the dose-limiting toxicity (DLT) observed at 20 MBq/kg bw and a dose expansion cohort is currently open for enrollment at 15 MBq/kg with HH1 pre-dosing. Arm 2 is currently open for enrollment. Tumour response was assessed by FDG PET/CT scans (Cheson 2007), and pts will be followed for 5 years. Results: Arm 1:A total of13 (M/F 11/2) pts, median age 68 years, follicular lymphoma (n=12), and mantle cell lymphoma (n=1) have been enrolled since the study start in December 2012. The range of prior therapies was 1 to 8, where 5 of 13 pts were refractory to rituximab. The most common toxicities observed were hematologic and all DLTs were reversible and manageable. At 20 MBq/kg (n=3) G 3/4 neutropenia and/or thrombocytopenia were observed in all pts and platelet transfusions were required in 2 pts. At 15 MBq/kg (n=6) DLTs were: 1 G 3 thrombocytopenia lasting >14 days and 1 G 4 neutropenia/ thrombocytopenia lasting >7 days. The median time to nadir for platelets and neutrophils was 40 and 49 days, respectively. No pts experienced febrile neutropenia. Serious AEs were reported in 5 pts: at 10 MBq/kg pneumonia (possibly related) and pulmonary embolism (PE) unrelated, in the same pt, with history of PE; thrombocytopenia requiring platelet transfusions (2 pts) and epistaxis in 1 of them (20 MBq/kg), possibly related; transient atrial fibrillation (2 pts) at 15 MBq/kg, possibly related. No secondary malignancies or other long term events have been observed. Best overall tumor response observed across all dose levels were 4 complete and 3 partial remissions, 2 stable disease and 4 progression of disease (one pt had confirmed transformed lymphoma at 3 months). The duration of response (complete and partial remissions) ranged from 6 to more than 21 months. One patient is still in remission after 2 years. The median response duration has not yet been reached. Arm 2: Inclusion in this arm is ongoing. Data on efficacy and safety will be presented and compared with the pts receiving pre-dosing. Conclusions: 177Lu-DOTA-HH1, which is a single dose ready-to-use formulation, has a predictable and manageable safety profile. Most AEs were hematological in nature, all transient and reversible. Promising efficacy and durable responses have been observed. 177Lu-DOTA-HH1 has the potential to be a novel therapy for B-cell malignancies. Disclosures Kolstad: Nordic Nanovector ASA: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bolstad:Nordic Nanovector ASA: Employment. Bruland:Nordic Nanovector ASA: Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership. Hartvig Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Germinal Center-Derived Lymphomas and Plasmacytomas in Mice with Targeted Deletion of MiR-15a/16-1 in Activated B Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 743-743
Author(s):  
Tomasz Sewastianik ◽  
Jianjun Zhao ◽  
Meng Jiang ◽  
Jianli Wang ◽  
Vinodh Pillai ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Plasma Cells ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Activated B Cells

Abstract MicroRNA (miR)-mediated gene regulation plays critical roles in B-cell development and dysregulated expression of miRs has been implicated in the pathogenesis of various types of B-cell malignancies. Somatic deletions of chromosome 13q14, harboring miR-15a/16-1, occurs frequently in B-cell lymphomas suggesting that members of this miR family are tumor suppressors. Consistently, mice with CD19-Cre-induced deletion of miR-15a/16-1 in early B-cells and follicular B-cells develop chronic lymphocytic leukemia (CLL). Since the 13q14 deletion is observed in a broader range of B-cell malignancies, we hypothesized that the type of B-cell malignancy resulting from miR-15a/16-1 down-regulation may depend on the stage of B-cell development at which this deletion occurs. Therefore, we generated a transgenic mouse model in which conditional deletion of miR-15a/16-1 takes place at later stages of B-cell development. To delete miR-15a/16-1 in activated B-cells, miR-15a/16-1fl/fl mice were mated with AID-Cre+/+ mice to obtain AID-Cre+/-; miR-15a/16-1fl/fl compound mice that expressed Cre recombinase from the Activation-induced Cytidine Deaminase (AID promoter) gene - a gene needed for generation of somatic hypermutations in the immunoglobulin (Ig) variable region (V) genes that is highly expressed in activated B-cells and is a well-known marker for germinal center (GC) B-cells. Expression levels of both miR-15a and miR-16-1, but not miR-15b were decreased in GC B-cells of AID-Cre+/-; miR-15a/16-1fl/fl mice as compared with control AID-Cre+/- mice when evaluated by In Situ Hybridization (ISH) analysis. Given that in humans miR-15a, b and 16 are also expressed in GC B-cells, these results demonstrate the validity of this mouse model in which the biological consequences of miR-15a/16-1 deletion can be studied. Next we assessed whether miR-15a/16-1 deletion could affect proliferation and/or survival of GC B-cells. GCs in the spleens of AID-Cre+/-; miR-15a/16-1fl/fl mice at 10 weeks of age were significantly increased in both number and size, and contained a larger number of Ki-67-positive B-cells as compared with spleens of AID-Cre+/- mice. No significant differences in the number of apoptotic cells, neither in the expression of the miR-15a/16-1 putative target BCL2 were detected, indicating that miR-15a/16-1 may play important roles in the proliferation, but not survival of GC B-cells. Apart from mild splenic enlargement and increased number and size of GCs, AID-Cre+/-, miR-15a/16-1fl/fl mice where indistinguishable from AID-Cre+/- mice between 8 and 40 weeks of age as assessed by weight and posture. However, after 48 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-, miR-15a/16-1fl/fl mice but none from control cohorts (0/30) showed signs of disease. Gross pathologic examination of euthanized AID-Cre+/-; miR-15a/16-1fl/fl mice revealed enlargement of the spleen and lymph nodes. Detailed histological examination revealed in most instances an effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells, or less commonly by sheets of plasma cells in interfollicular areas. Two distinct patterns of B220+BCL6+BCL2- B-cell lymphomas were identified after detailed analysis. The most common (47%) resembled human follicular lymphoma (FL) and the next in frequency (28%) resembled human diffuse large B-cell lymphoma (DLBCL). The other group of tumors (25%) resembled human plasmacytoma (PC). All three tumor subtypes were clonal, hypermutated and associated with different degrees of preservation of the dendritic meshwork in the lymph nodes. The comparison of lymphomas arising in AID-Cre+/-; miR-15a/16-1fl/fl mice and CD19-Cre+/-; miR-15a/16-1fl/fl mice corroborated that deletion of miR-15a/16-1 at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated miR-15a/16-1 expression in human FL and PC and showed that miR-15a/16-1 abundance is significantly decreased in those malignancies when compared with nodal B-cells in reactive GCs and normal plasma cells in interfollicular areas respectively, suggesting that miR-15a/16-1 may play important roles in normal GC B-cell development as well as in the pathogenesis of FL and PC in humans. Disclosures Ghobrial: BMS: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

scholarly journals IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1325-1325 ◽  
Author(s):  
Jae-Woong Lee ◽  
Huimin Geng ◽  
Zhengshan Chen ◽  
Eugene PARK ◽  
Lars Klemm ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Transmembrane Protein ◽  
Cell Receptor ◽  
Surface Expression ◽  
Akt Signaling ◽  
Mrna Levels ◽  
Surface Receptor

Abstract Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells. Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes. Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts. Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4420-4420 ◽  
Author(s):  
Shih-Shih Chen ◽  
Jeffery Lorne Kutok ◽  
Gerardo Ferrer ◽  
Priyadarshini Ravichandran ◽  
Michael Ibrahim ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Supporting Cells ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Ibrutinib Resistance

Abstract Novel agents targeting the B-cell receptor signaling pathway, such as ibrutinib and idelalisib, are effective in patients with chronic lymphocytic leukemia (CLL), but complete responses are infrequent and residual disease often remains. Patients may discontinue ibrutinib because of unacceptable adverse events or if they develop ibrutinib resistance due to Bruton tyrosine kinase (BTK) or phospholipase C gamma 2 (PLCG2) mutations or other less-well-defined mechanisms. Therefore, alternative therapies that can overcome ibrutinib intolerance and resistance are needed. Duvelisib (DUV) is an oral dual phosphatidylinositol 3-kinase (PI3K)-δ and -γ inhibitor with activity in patients with relapsed/refractory CLL and a manageable safety profile. Preclinical studies point to distinct roles of PI3K-δ and -γ in CLL biology, suggesting that dual isoform inhibition may enhance efficacy by targeting both CLL and CLL-supporting cells. Importantly, DUV potently inhibits PI3K-δ and -γ in human peripheral blood mononuclear cells and CLL cells. Here, we further characterize the distinct functions of PI3K-δ and -γ in CLL and examine the mechanisms of action of DUV in vitro and in a patient-derived xenograft (PDX) model, highlighting the ability of DUV to overcome ibrutinib resistance. We first assessed the in vivo efficacy of PI3K-δ and/or PI3K-γ inhibition on CLL B cells using a PDX mouse model in which activated patient-derived T cells and CLL B cells were injected into alymphoid NOD-scid IL-2Rγnull (NSG) mice. Cells from 4 patients were transferred and allowed to expand for 2 weeks, after which mice were treated with DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor for 3 weeks. DUV and the PI3K-δ inhibitor, but not the PI3K-γ inhibitor, significantly decreased the number of CLL B cells in the spleens of mice. In another set of experiments, combination treatment with the PI3K-δ and -γ inhibitors more potently impaired CLL B-cell survival than the PI3K-δ inhibitor alone. Next, the effects of PI3K-δ or -γ inhibition on CLL-supporting cells, ie, autologous T cells and murine macrophages, were examined in the PDX model using the same 4 patient samples. Significant decreases in both patient-derived T cells and murine macrophages were observed in the spleens of mice treated with DUV or the PI3K-γ inhibitor but not with the PI3K-δ inhibitor. Therefore, the function of PI3K-δ and -γ in macrophages and macrophage-supported CLL cell survival were examined in vitro. Murine bone marrow-derived macrophages were polarized via interleukin 4 and macrophage colony stimulating factor in the presence of DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor. Both DUV and the PI3K-γ inhibitor impaired M2 polarization, assessed by arginase 1 (ARG1) mRNA expression. Culture with M2 macrophages increased CLL B-cell viability, and the addition of DUV inhibited this survival-promoting activity more than the PI3K-δ or -γ inhibitors alone. To assess whether DUV could inhibit CLL B cells from ibrutinib-unresponsive patients, activated T cells and CLL B cells from 2 patients who progressed on ibrutinib (1 with a BTK C481S mutation and 1 without a BTK mutation) were transferred into NSG mice and allowed to expand for 2 weeks, and then mice were treated with DUV or ibrutinib for 3 weeks. For both patient samples, there was a > 10-fold reduction in the number of CLL B cells recovered from the spleens of mice treated with DUV, as well as a significant reduction in the percentage of proliferating CLL B cells. In contrast, ibrutinib did not have significant impact on CLL B-cell numbers or proliferation in the spleen. In conclusion, DUV inhibits the in vivo survival and proliferation of leukemic B cells from CLL patients, including those who have progressed on ibrutinib. Dual PI3K-δ and -γ inhibition is more effective at inhibiting CLL B cells in vivo than PI3K-δ inhibition alone. Moreover, PI3K-γ inhibition shifts macrophage polarization away from a CLL-supportive M2 phenotype. Thus, DUV exerts inhibitory effects on CLL B cells and on CLL-supporting T and myeloid cells. Overall, these findings elucidate the non-redundant roles of PI3K-δ and -γ in CLL and demonstrate the potent antitumor activity of dual PI3K isoform inhibition by DUV in ibrutinib-resistant patient CLL cells in vivo. Further investigation of DUV as a therapeutic option for patients who are refractory to or intolerant of ibrutinib or other BTK inhibitors is ongoing in a phase 2 clinical trial (BRIO; NCT03370185). Disclosures Chen: Janssen: Research Funding; ArgenX: Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Kutok:Infinity Pharmaceuticals: Employment, Equity Ownership. Barrientos:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Weaver:Verastem Oncology: Employment, Other: Stockholder; Agios Pharmaceuticals: Employment; Femto Dx: Equity Ownership. Pachter:Verastem: Employment, Other: Stockholder. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

scholarly journals IFITM3 (CD225) Regulates CD19 Surface Expression and CD19-Mediated Activation of PI3K Signaling in Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1070-1070
Author(s):  
Jae-Woong Lee ◽  
Huimin Geng ◽  
Zhengshan Chen ◽  
Eugene Park ◽  
Angela Park ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
Akt Signaling ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
Wild Type ◽  
Cycle Arrest ◽  
Cd19 Expression

Abstract Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21 on mouse and human B cells. However, besides its interaction with CD19, specific functions of Ifitm3 in normal B cells development and, potentially leukemogenesis, are not known. Recent approaches based on CD19-specific chimeric antigen receptors (CAR) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (e.g. Grupp et al., N Engl J Med 2013). However, in some cases, CD19 CAR treatment was followed by ALL relapse developing from a clone that lacked CD19 surface expression. Therefore, we studied factors that can potentially regulate CD19 surface on normal and leukemic pre-B cells. Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in near-complete loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. Reconstitution of IFITM3 in patient-derived pre-B ALL cells rescued both CD19 expression and increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT, pointing to a central role of IFITM3 in regulating CD19/PI3K-AKT signaling in pre-B cells and pre-B ALL. Deletion of Ifitm3 also resulted in significant cell cycle arrest in the G0/G1 phase (P<0.001) and increased protein levels of p53 and p21 compared to wild-type pre-B ALL cells. Moreover, treatment with low dose adriamycin (25 ng ml-1) for induction of modest DNA damage significantly induced cellular senescence in Ifitm3-/- but not wild-type pre-B ALL cells. Consistent with Ifitm3-mediated proliferative defects of Ph+ ALL cells, Ifitm3-/- Ph+ ALL cells exhibited reduced self-renewal capacity (P=0.0004) decreased colony in colony forming assay compared to wild-type cells. Furthermore, Ifitm3 deficient B cell progenitors also showed significant inhibition of proliferation with inhibition of phosphorylation of Stat5Y694and c-Myc expression. 4-hydroxytamoxifen (4-OHT)-inducible activation of CD19 rescued proliferative defects in Ifitm3 deficient B cell progenitors with release from G0/G1 cell cycle arrest associated with upregulation of phosphorylation of Stat5 Y694 and c-Myc expression. In addition, we found that CD19 positively regulates the surface expression of IL7R in B cell progenitors. Interestingly, CD19low population in Ifitm3 deficient B cell progenitors showed lower surface expression of IL7R compared to CD19high population. In contrast to B cell progenitors, forced expression of CD19 did not increase the proliferation of wild-type Ph+ ALL cells. Surprisingly, upregulation of CD19 induced apoptosis in Ifitm3-deficient Ph+ ALL cells with significant inhibition of BCR-ABL1 activity, phosphorylation of Stat5Y694and Bcl2 expression. Conclusion: These findings identify novel role of IFITM3 in regulating CD19 surface expression and CD19-Y513 mediated PI3K-AKT signaling in pre-B cells and pre-B ALL. Preliminary experiment showed that agonistic antibodies against IFITM3/CD225 increased CD19/PI3K-AKT signaling and proliferation in pre-B ALL cells and future studies will elucidate whether blocking antibodies (e.g. decoy Fc-fusion molecules) will have useful effects in targeting CD19/PI3K-AKT signaling in human pre-B ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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