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 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.

scholarly journals Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

2002 ◽  
Vol 9 (2) ◽  
pp. 86-95 ◽  
Author(s):  
Denise A. Kaminski ◽  
John J. Letterio ◽  
Peter D. Burrows
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
B Cell ◽  
Transforming Growth Factor ◽  
Plasma Cells ◽  
Population Analysis ◽  
Cell Development ◽  
B Cell Development

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

scholarly journals Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development

2016 ◽  
Vol 113 (32) ◽  
pp. 9063-9068 ◽  
Author(s):  
Nilushi S. De Silva ◽  
Michael M. Anderson ◽  
Amanda Carette ◽  
Kathryn Silva ◽  
Nicole Heise ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Alternative Pathway ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Surface Receptor ◽  
Signaling Cascade

The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-5
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
Cell Immune Response ◽  
Germinal Center Hyperplasia

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

BCR-ABL1-Positive Acute Lymphoblastic Leukemia Patients Treated with Only TKI Vs Conventional Chemo Plus TKI Therapy Show Similar DNA Alterations At Relapse Targeting Key Regulators of Tumor Suppression, Cell Cycle Control, and Lymphoid/B-Cell Development,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3580-3580
Author(s):  
Ilaria Iacobucci ◽  
Heike Pfifer ◽  
Annalisa Lonetti ◽  
Cristina Papayannidis ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Board Of Directors ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Cell Cycle Control ◽  
Cell Development ◽  
B Cell Development ◽  
Advisory Committees ◽  
Copy Number Changes

Abstract Abstract 3580 Introduction: Although treatment with tyrosine kinase inhibitors (TKIs) has revolutionized the management of adult patients with BCR-ABL1 -positive acute lymphoblastic leukemia (ALL) and significantly improved response rates, relapse is still an expected and early event in the majority of them. It is usually attributed to the emergence of resistant clones with mutations in BCR-ABL1 kinase domain or to BCR-ABL1 -independent pathways but many questions remain unresolved about the genetic abnormalities responsible for relapse after TKI and chemotherapy-based regimens. Patients and methods: In an attempt to better understand the genetic mechanisms responsible for this phenomenon, we have analyzed matched diagnosis-relapse samples from 30 adult BCR-ABL1 -positive ALL patients using high resolution Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI, n=15 pairs and Genome-Wide Human SNP 6.0, n= 15 pairs). Genetic differences were analyzed in terms of copy number changes and loss of heterozygosity (LOH) events. 20 patients were enrolled in clinical trials of GIMEMA AL Working Party and treated with imatinib alone or in combination with conventional chemotherapy (40%) or dasatinib as frontline therapy (60%). The median age at diagnosis was 54 years (range 23–74) and the median blast cell count was 97% (range 60–99). The median time to relapse was 27 months (range, 9–104). 10 patients were treated according to the GMALL trials, a high-dose chemotherapy based protocol in combination with imatinib. The median age at diagnosis was 65 years (range 19–79) and the median leucocyte count was 37300/μl (range 5000 – 220000/μl). The median time to relapse was 9.8 months (range, 3 – 25). Results: First, we compared diagnosis and relapse samples for the presence of macroscopic (> 1.5 MB) copy number alterations (CNA). Novel acquired macroscopic CNAs were detected in 7/20 (35%) TKI relapse cases and included losses of 3p12-p14, 5q34, 9q34, 10q24 and 12p13-p12 and gains of 1q, 9q34-q33 and 22q and in 4/10 (40%) chemotherapy-relapse cases and included losses of 9p21 and 12q21–22 and gains of all chromosome 8 or part of it in 2 patients. Since no common patterns of acquired alterations were observed, it is likely that relapse may be due to a more generalized genetic instability rather than to specific mechanisms. Moreover, chemotherapy did not select resistant clones with higher number of alterations. 8/20 (40%) TKI resistant cases and 4/10 chemotherapy resistant patients harbored the same CNAs present in the matched diagnosis sample (losses of 9p21 in 7 cases, 7p and 22q11 in single cases and gains of chromosomes 1q, 4, 8q, 17q and 21), indicating a common clonal origin. In contrast, in 5/20 (25%) TKI resistant cases and 4/10 (40%) chemotherapy resistant patients macroscopic CNAs present at diagnosis were lost at relapse (losses of chromosomes 7, 11q, 14q, 15q, 16q and 19p and gains of 5q, 8q, 9q34 and 22q11). Thereafter, we compared diagnosis and relapse samples for microscopic CNAs (< 1.5 MB). The alteration most frequently acquired at relapse was loss of the tumor suppressor CDKN2A (53% vs 33 % of diagnosis). Other common acquired CNAs at relapse included gains of ABC transporter genes, such as ABCC1, ABCC6 (1q41) and BCL8 (15q11); losses affected EBF1 (5q33) and IGLL3 (22q11) genes involved in B-cell development, BTG1 (12q21) involved in cell cycle regulation and CHEK2 (22q12) involved in DNA repair. Interestingly, for all relapse cases analysis of IKZF1 deletions, identified in 80% of patients, demonstrated a clonal relationship between diagnostic and relapse samples, suggesting that this alteration is not acquired with relapse but it is maintained with fidelity from diagnosis working as a marker of disease. The majority (92%) of relapse samples harbored at least some of the CNAs present in the matched diagnosis sample, indicating a common clonal origin. Conclusions: Genomic copy number changes evolving from diagnosis to relapse have been identified demonstrating that a diversity of alterations contributes to relapse and with the most common alterations targeting key regulators of tumor suppression, cell cycle control, and lymphoid/B cell development. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, Ateneo RFO grants, PIO program, Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Ottmann:Novartis Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Pathological Roles of p18 Deficiency Toward B Cell Malignances

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2448-2448
Author(s):  
Sha Hao ◽  
Fang Dong ◽  
Wen Zhou ◽  
Hui Cheng ◽  
Shihui Ma ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Poor Prognosis ◽  
Absolute Number ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Malignancies ◽  
Hematopoietic Stem ◽  
Hematopoietic Malignancies ◽  
Low Expression

Abstract The cyclin-dependent kinases inhibitor CDKN2C (p18INK4C, p18) is a member of the INK4 family that specifically blocks the activity of CDK4/6 in the G1 phase of cell cycle. In the hematopoietic system, deletion of p18 was indicated to be associated with T cell malignancies in mice and B cell malignancies in humans. Moreover, p18 deficiency is a poor prognosis factor for the patients with multiple myeloma (MM). However, a formal investigation on the pathological roles of p18 deficiency in hematopoietic malignancies, especially B cell malignancies is lacking. In this study, we first obtained direct clinical relevance of p18 deficiency with hematopoietic malignancies. Based on the Oncomine data set analysis, low expression of p18 was found in the patients with B-Cell Acute Lymphoblastic Leukemia (54 out of 80). In addition, by Gene expression Profile (GEP) analysis (n=361) and multi-color FISH analysis (n=265) of first-visit MM patients, there were 11% MM patients showed low expression and 9.06% biallelic deletion of p18 gene respectively, which was correlated with poor prognosis. Further analysis indicated higher expression of c-Myc, Bcl-2 and TRAF3 in p18-deleted MM patients or MM cell lines. We then focused on the impact of p18 deletion on B cell development with the mice deficient in p18 (p18-/-). The frequency and absolute number of B220+ B cell were significantly decreased in the bone marrow (21.075±0.168% vs 13.956±1.613%, n=5) or spleen (49.320±1.773 vs 35.35±1.673, n=5) of p18-/- mice. Secretion of immunoglobulin (Ig) from plasma cells was also impaired. Furthermore, p18-/- BM or enriched hematopoietic stem cell (LSK+) transplantation also recaptured the deficiency of mature B cells in the recipients despite higher repopulation in the p18-/- group. Ectopic over-expression of p18 in the hematopoietic stem and progenitor cells (HSPCs) via retroviral transduction could partially correct the abnormality of p18-/- B cells in the transplant recipients. These results suggested that the defect of B cell development in the absence of p18 was intrinsic to the hematopoietic cells, rather than extrinsic (via micro-environmental). To further define the effects of p18 deficiency on HSPCs prior to B cell commitment, we enumerated the frequencies of LT-HSC, MPP, CMP, GMP, MEP, Lin-IL-7R+ and CLP cell populations in p18-/- or control mice. There was no significant difference in the frequency or absolute number of CMP, GMP, MEP, or CLP between p18-/- and control groups. Notably however, the colony-forming cells of pre-B cells in p18-/- BM were significantly increased (24.4±2.1 vs 32.6±1.8, n=5). Moreover, we also examined the B cells at different developmental stages including pre-pro-B cell, pre-B, immature-B and mature B cells in BM, as well as transitional stage 1(T1), transitional stage 2 (T2) and mature B cells in the spleen. Our data showed an accumulation of the cells at pre-B cell stage in the absence of p18, while dramatically decreased at mature B cells stage. To further explore the molecular basis, single cell RT qPCR analysis was performed and revealed that the transcription factors including Foxo1, Rag2, E2A, EBF1and Pax5 were significantly higher inCLP, pro-B, pre-B, immature-B subpopulations of p18-/- group. However, lamada 5, which is necessary for B cells maturation, was remarkably decreased in p18-/- immature B cells compared with control group. Taken together, our study provides definitive evidence for the disruption of B cell development due to p18 deficiency and this new evidence underlies the pathological contributions of p18 down-regulation or deletion to B cell malignancies in humans. Disclosures No relevant conflicts of interest to declare.

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.

Tumor Suppressors BTG1 and BTG2 Fulfill Both Unique and Overlapping Functions During Normal B Lymphocyte Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1303-1303
Author(s):  
Esther J.H. Tijchon ◽  
Liesbeth van Emst ◽  
Jørn Havinga ◽  
Jean-Pierre Rouault ◽  
Felice Tirone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Tumor Suppressors ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
B Cell Malignancies ◽  
Cell Stage

Abstract Abstract 1303 B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common form of cancer in children, characterized by genetic aberrations affecting master regulators of lymphoid differentiation, such as RUNX1, IKZF1, TCF3, and PAX5, as well as tumor suppressor genes that control the cell cycle, including RB1 and CDKN2A. Another gene frequently altered in BCP-ALL is BTG1, which displays highly clustered mono-allelic deletions in childhood BCP-ALL (9%) and adult ALL (6%). The frequency of BTG1 deletions is two- to three-fold higher in ETV6-RUNX1- and BCR-ABL1-positive leukemias. BTG1, and its close homologue BTG2 regulate gene expression, for instance by associating with protein arginine methyltransferase 1 (PRMT1), affecting the activity of a variety of transcription factors, including several nuclear hormone receptors and HoxB9. In addition, BTG1 and BTG2 have been implicated in regulating mRNA stability by interacting with the Ccr4-Not complex. Recent studies have also identified missense point mutations in BTG1 and BTG2 in about 20% of non-Hodgkin lymphomas, arguing that altered function of these genes contributes to B cell malignancies. To investigate a role of BTG1 and BTG2 in B cell development, we studied the phenotype of Btg1 and Btg2 single knockout (KO) and Btg1;Btg2 double KO mice. Animals deficient for either BTG1 or BTG2 displayed a mild B cell phenotype with a moderate reduction of ∼20% in the total amount of B220+ progenitor B cells in bone marrow, while splenic B cells were present at normal frequencies. More detailed analyses revealed that Btg1−/− and Btg2−/− mice both showed a partial block at the pre-pro-B cell stage (Hardy fraction A). Methylcellulose colony assays in the presence of interleukin-7 (IL-7) demonstrated 30% fewer colonies using bone marrow from Btg2−/− mice, whereas 70% fewer colonies were obtained using bone marrow derived from Btg1−/− mice. To assess whether BTG1 and BTG2 fulfill redundant functions during B cell development, we analyzed the phenotype of Btg1−/−;Btg2−/− mice. Hence we observed that the combined loss of BTG1 and BTG2 led to a much stronger block in B cell differentiation, with the majority of progenitor B cells arrested at the pre-pro-B cell stage. In the spleens of these double knockout mice we observed a roughly 50% reduction in B220+ IgM+ B cells, suggesting that these genes act to modify the activity of B lineage transcription factors rather than to fully block their activities. This is consistent with a role for these genes as modifiers of transcriptional activity. Current studies are aimed at defining the molecular targets regulated by BTG1 and BTG2 during early B cell development using RNA sequencing and protein interaction experiments. In conclusion, our data demonstrate that BTG1 and BTG2 act as important regulators of normal B cell differentiation, and that this function might be critical for their role as tumor suppressors in (early) B cell malignancies. Disclosures: No relevant conflicts of interest to declare.

Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4629-4639 ◽  
Author(s):  
Nancy D. Borson ◽  
Martha Q. Lacy ◽  
Peter J. Wettstein
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Mrna Expression ◽  
Healthy Subjects ◽  
Plasma Cells ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Populations ◽  
Expression Levels

Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell development. We encountered an unidentified isoform of B cell–specific activator protein (BSAP, or Pax5) in MM cells while performing differential analyses to compare mRNA expression in malignant and normal plasma cells. Pax5 is a transcription factor that plays a central role throughout B-cell development until the point of terminal differentiation. Our finding of this unique isoform prompted us to investigate Pax5 isoform usage in plasma cells and B-cell populations in other MM and healthy subjects. In contrast to normal Pax5 expression, we observed multiple isoforms of Pax5 in conjunction with low levels of expression of the full-length Pax5 in B cells from MM patients. The expressed isoforms in MM varied considerably from patient to patient, with no clear pattern. We also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte–induced maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma cells in MM.

scholarly journals In Vitro-Selected Nanobody-Based Cellular Therapy Targeting CD72 for Treatment of Refractory B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1337-1337
Author(s):  
Matthew Nix ◽  
Yu-Hsiu T. Lin ◽  
Huimin Geng ◽  
Makeba Marcoulis ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Board Of Directors ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified &gt;900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's &lt; 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents & Royalties. 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.

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