Co-Expression of SYK and ZAP70 Subverts Negative B-Cell Selection and Enables Oncogenic Signaling in Multiple B-Cell Malignancies

B-cells are under intense selective pressure to eliminate autoreactive or premalignant clones. B-cell receptor (BCR) signals are required for survival, however, BCR-signaling exceeding maximum thresholds often reflects signaling from an autoreactive BCR or a transforming oncogene and triggers negative selection and cell death. The tyrosine kinase SYK initiates BCR-downstream signaling in B-cells while its close relative ZAP70 is almost exclusively expressed in T-cells. Interestingly, the segregation of SYK to B-cells and ZAP70 to T-cells is less confined in malignant lymphopoiesis suggesting that the balance of these related kinases may alter signaling output in disease and contribute to development of leukemia. As previously shown in B-cell chronic lymphocytic leukemia (B-CLL), we identified aberrant ZAP70 expression as a frequent feature in multiple other B-cell malignancies that depend on survival signals from a functional (pre-) BCR (E2A-PBX1+ pre-B ALL, and mantle cell lymphoma) or harbor oncogenic mimics of the BCR (BCR-ABL1+ B-ALL). Studying SYK and ZAP70 expression by single-cell Western blot, co-expression of the two tyrosine kinases was extremely rare in normal B- and T-cell populations. In contrast, >50% of tumor B-cells in mantle cell lymphoma, pre-B ALL and CLL co-expressed SYK and ZAP70. Despite their structural similarities, genetic deletion and engineered reconstitution of SYK and ZAP70 in human B-cell lymphoma cells revealed striking functional differences. Proximity-dependent biotin identification (BioID) analyses identified that SYK, but not ZAP70, engaged the PI3K pathway via interaction with CD19. Consistent with this, reconstitution with SYK and SYK-ZAP70 but not ZAP70 alone promoted survival and proliferation. Detailed analysis of BCR-mediated cascades in lymphoma cells expressing SYK, ZAP70 or SYK-ZAP70 established that ZAP70 is only weakly efficient at propagating BCR-mediated calcium and downstream pathway activation in B-cells. Strikingly, co-expression of ZAP70 with SYK resulted in re-wired BCR-signaling of intermediate strength: compared to cells expressing only SYK, SYK-ZAP70 co-expressing cells had markedly reduced activation of the BLNK-BTK-PLCγ pathway, further reflected in BCR-induced Ca2+ signaling with delayed onset, lower amplitude but longer duration. In this way, we speculated that SYK and ZAP70 may be present within close proximity at the apex of BCR-initiated interactions, and hence compete for downstream substrates resulting in a re-wiring of classic signaling programs propagated normally by SYK. To explore this, we utilized proximity ligation assays (PLA) to monitor the proximity of SYK and ZAP70 in resting or BCR-stimulated B-cells, and found that SYK and ZAP70 co-exist within close proximity consistent with the view that varying levels of these kinases may alter B-cell signaling output. Functional experiments further showed that phosphomimetic activation of SYK, but not ZAP70, induced hyperactivation of PI3K-signaling and acute BTK-mediated cell death in pre-B ALL cells. In line with altered BCR-signaling strength and quality in SYK and ZAP70 co-expressing cells, over-expression of Zap70 in pre-B ALL cells rescued auto-immune checkpoint activation induced by hyper-activation of BCR-associated signaling. To study functional consequences of SYK-ZAP70 co-expression during normal B-cell development, we generated a novel knock in Zap-70+/Mb1-Cre+mouse model, to induce conditional expression of Zap70 in the B cell compartment from the proB stage. Consistent with compromised central tolerance checkpoints, Syk-Zap70 co-expressing pro/pre-B and immature B-cells had reduced spontaneous apoptosis rates and gave rise to autoantibody production against multiple self-antigens. Importantly, our findings highlight a previously unrecognized role for ZAP70 in oncogenic BCR-signaling and we conclude that the co-expression of ZAP70 mitigates the ability of SYK, downstream of an autoreactive BCR or a transforming oncogene, to trigger negative B-cell selection and cell death (Figure 1). Disclosures Weinstock: Celgene: Research Funding. Meffre:AbbVie: Consultancy, Other: Grant.

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The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

Blood ◽

10.1182/blood-2010-05-283309 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4532-4541 ◽

Cited By ~ 164

Author(s):

Michael Hudecek ◽

Thomas M. Schmitt ◽

Sivasubramanian Baskar ◽

Maria Teresa Lupo-Stanghellini ◽

Tetsuya Nishida ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Chimeric Antigen Receptor ◽

Cell Lymphoma ◽

Embryonic Stem ◽

Antigen Receptor ◽

Orphan Receptor ◽

Mantle Cell

Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

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Potent B-Cell Depletion by IMGN529, a CD37-Targeting Antibody-Maytansinoid Conjugate for the Treatment of B-Cell Malignancies,

Blood ◽

10.1182/blood.v118.21.3726.3726 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3726-3726

Author(s):

Jutta Deckert ◽

Sharon Chicklas ◽

Yong Yi ◽

Min Li ◽

Jan Pinkas ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Whole Blood ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Depletion ◽

B Cell Depletion ◽

B Cell Malignancies

Abstract Abstract 3726 CD37 is a B-cell surface antigen which is widely expressed on malignant B cells in non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues CD37 expression is limited to blood cells and lymphoid tissues. This restricted expression profile makes CD37 an attractive therapeutic target for antibodies and antibody-drug conjugates. We developed a novel anti-CD37 antibody, K7153A, which provides a unique combination of functional properties: it demonstrated strong pro-apoptotic and direct cell killing activity against NHL cell lines and could mediate effector activity such as CDC and ADCC. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. The direct cytotoxic potency of the K7153A antibody was superior to that of the CD20-directed rituximab and was further enhanced with maytansinoid conjugation in IMGN529. In vivo, IMGN529 demonstrated better anti-tumor activity than the K7153A antibody in established subcutaneous follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and CLL xenograft models in SCID mice. A single administration of IMGN529 showed similar or improved efficacy compared to anti-CD20 antibodies or standard chemotherapy where tested. Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissue sections was performed to evaluate CD37 expression. CD37 exhibited a similar prevalence to CD20 in subtypes of NHL such as FL, DLBCL, Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL). B-cell depletion is an important measure of efficacy for targeted therapies, such as CD20-directed antibodies, in B-cell malignancies. CD37 expression in blood cells from healthy human donors was measured by quantitative flow cytometry in comparison to CD20. The greatest CD37 expression was found in B cells at approximately 77,000 antibodies bound per cell (ABC), which was similar to CD20 expression in B cells at 95,000 ABC. In other blood cell types CD37 staining was seen at low levels, about 2,000 – 5,000 ABC, in monocytes, NK cells and T cells. In vitro depletion experiments were performed with purified peripheral blood mononuclear cells (PBMCs) and with whole blood, both derived from several healthy donors. Cells were incubated for 1 hr with 10 μg/mL of either K7153A, IMGN529, CD37-targeting TRU-016, rituximab or the anti-CD52 antibody alemtuzumab, with cell depletion determined relative to counting beads by flow cytometry. The K7153A antibody and the IMGN529 conjugate efficiently and specifically depleted B-cells in a dose-dependent manner in the context of purified PBMCs and whole blood. With purified PBMCs, both K7153A and IMGN529 caused 50–60% depletion of B cells, with little to no depletion of T cells or monocytes. IMGN529 was more potent than rituximab, which led to 30–40% B-cell depletion, or TRU-016, which caused 20–30% B-cell depletion. IMGN529 also was more specific than alemtuzumab, which depleted T-cells and monocytes as well as B cells. With whole blood samples, both K7153A and IMGN529 resulted in 30–40% B-cell depletion with no effect on T cells, NK cells or monocytes. IMGN529 was again more potent than rituximab or TRU-016, which caused approximately 10% B-cell depletion, and was more specific than alemtuzumab, which depleted the majority of T cells in addition to 40% of B cells. IMGN529 embodies a unique B-cell targeted agent as it combines the intrinsic pro-apoptotic, CDC and ADCC activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by the targeted delivery of its maytansinoid payload. It is highly active in vitro and in vivo against B-cell lymphoma and CLL cell lines. In addition, it mediates specific B-cell depletion in vitro that is greater than B-cell depletion by CD20-directed rituximab. Together, these findings indicate that IMGN529 is a promising therapeutic candidate for the treatment of B-cell malignancies. Disclosures: Deckert: ImmunoGen, Inc.: Employment. Chicklas:ImmunoGen, Inc.: Employment. Yi:ImmunoGen, Inc.: Employment. Li:ImmunoGen, Inc.: Employment. Pinkas:ImmunoGen, Inc.: Employment. Chittenden:ImmunoGen, Inc.: Employment. Lutz:ImmunoGen, Inc.: Employment. Park:ImmunoGen, Inc.: Employment.

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A Phase I/II Study of Vorinostat (SAHA), Cladribine (2-CdA), and Rituximab Shows Significant Activity in Previously Untreated Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v118.21.441.441 ◽

2011 ◽

Vol 118 (21) ◽

pp. 441-441 ◽

Cited By ~ 3

Author(s):

Stephen Spurgeon ◽

Andy I Chen ◽

Craig Okada ◽

Samir Parekh ◽

Violetta V. Leshchenko ◽

...

Keyword(s):

Cell Death ◽

Phase I ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Dna Hypomethylation ◽

Tumor Cell Death ◽

B Cell Malignancies ◽

Mantle Cell ◽

Correlative Studies

Abstract Abstract 441 Background: Despite significant progress in the treatment of mantle cell lymphoma (MCL), relapse remains the norm and additional therapies are needed especially for patients who are not candidates for aggressive treatment approaches. Increasingly, it has become evident that epigenetic modifications, including DNA hypomethylation and histone deacetylase inhibition, are critical to the pathogenesis and treatment of hematologic malignancies; important to cancer biology; and may be essential to the development of treatment resistance in B-cell malignancies. Further development and understanding of new and effective treatment regimens that target the epigenome are needed. 2-CdA has activity in a variety of B and T cell malignancies. In addition to its cytotoxic effects, our preliminary work shows that 2-CdA has hypomethylating properties in lymphoid malignancies. When primary MCL and CLL cells -before and 96 hours after cladribine treatment-were analyzed by HELP (HpaII tiny fragment Enrichment by Ligation mediated PCR), an array based genome-wide methylation assay, 2-CdA affected DNA hypomethylation. One of the genes hypomethylated was identified as DUSP2, a dual specificity phosphatase gene that is a p53 target gene. DUSP2 dephosphorylates phosphoserine/threonine and phosphotyrosine residues, negatively regulating mitogen-activated protein (MAP) kinases ERK1 and ERK2, which are associated with cellular proliferation and differentiation in B-NHL. Vorinostat (SAHA) is a histone deacetylase inhibitor (HDACi), which has shown modest single agent activity in lymphoma and is FDA approved for use in cutaneous T cell lymphoma (CTCL). MCL cell lines treated with cladribine activated DUSP2 mRNA and when treated with the HDAC inhibitor SAHA synergistically increased transcription of DUSP mRNA. Furthermore, MCL treated with cladribine in vitro showed inhibition of global histone methylation. Our hypothesis is that cladribine and vorinostat synergistically activate silenced genes such as but not limited to DUSP 1 and 2 that are important for tumor cell death. The mechanism of rapid tumor cell death is under investigation, and does not appear to involve the classical apoptosis pathway. Given the need for novel therapies and the potential synergy seen with 2-CdA and SAHA, we initiated a Phase I/II trial combining SAHA, 2-CdA, and rituximab (SCR) for the treatment of B-cell non-Hodgkin's Lymphoma (NHL). The Phase I portion has been completed while Phase II is actively enrolling patients including those with newly diagnosed MCL. Methods: Phase I enrolled 10 patients with relapsed/refractory NHL. The MTD of vorinostat for the Phase I was 400 mg (D 1–14) combined with 2-CdA 5mg/m2 IV (D 1–5), and R 375 mg/m2 IV (weekly × 4 for cycle 1 and 1x/month) every 28 days for up to 6 cycles. Phase II eligibility includes relapsed NHL as well as previously untreated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Primary outcome is response rate (ORR). Scientific correlatives include analysis of CD20 expression, histone acetylation, gene microarray and HELP methylation analysis, ERK phosphorylation, and Q-PCR of potential target genes. Results: 52 patients (Phase I/II) have been enrolled and 45 patients have been treated. The ORR in evaluable relapsed patients (3 DLBCL, 10 MCL, 1 FL, 1 MZL, 7 CLL) is 32% (7/22). Among these relapsed patients, complete remissions (CR) have been observed in MCL as well as follicular and marginal zone lymphomas. Of the 20 previously untreated MCL patients, 19 have completed ≥ 2 cycles and are evaluable for response. ORR is 100% (19/19) with 79% (15/19) CR. Toxicities by CTCAE 3.0 criteria have primarily included reversible myelosuppression, fatigue, dehydration, 1 gr. 4 thrombo-embolic event (probably related), and 1 grade 5 pulmonary hemorrhage in a patient with relapsed pulmonary lymphoma. One previously untreated mantle cell lymphoma patient has ongoing Gr. 3 thrombocytopenia six weeks after completing therapy. Preliminary analysis of ongoing correlative studies is available in 1 MCL patient and shows DUSP2 upregulation. Conclusions: The SCR regimen shows activity across a number of B-cell malignancies and shows particular therapeutic promise in patients with previously untreated mantle cell lymphoma. Correlative studies are ongoing and will be presented. Future studies should continue to explore this regimen in previously untreated mantle cell lymphoma. Disclosures: Off Label Use: vorinostat (SAHA) is not FDA approved for the treatment of B cell lymphomas. Okada:Merck: Speakers Bureau. Epner:Merck: Speakers Bureau.

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A New Platform For Trivalent Bispecific Antibodies Used For T-Cell Redirected Killing Of B-Cell Malignancies

Blood ◽

10.1182/blood.v122.21.3078.3078 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3078-3078

Author(s):

Diane L Rossi ◽

Edmund A Rossi ◽

David M Goldenberg ◽

Chien-Hsing Chang

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

Tumor Cells ◽

Stock Option ◽

B Cell Malignancies ◽

Hla Dr ◽

Close Proximity

Abstract Background Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise both pre-clinically and clinically. The scFv-based constructs, including BiTE and DART, which bind monovalently to CD3 on T cells and to the target antigen on tumor cells, exhibit fast blood clearance and neurological toxicity due to their small size (∼55 kDa). Herein, we describe the generation of novel T-cell redirecting trivalent bsAbs comprising an anti-CD3 scFv covalently conjugated to a stabilized F(ab)2. The design was initially characterized with a prototype construct designated (19)-3s, which specifically targets CD19 on B cells. A panel of trivalent bsAbs was evaluated for their potential use in targeted T-cell immunotherapy of various B-cell malignancies. Potential advantages of this design include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. Methods The DOCK-AND-LOCKTM (DNLTM) method was used to generate a panel of B-cell targeting bsAbs, (19)-3s, (20)-3s, (22)-3s, and (C2)-3s, which target CD19, CD20, CD22, and HLA-DR, respectively. This was achieved by combining a stabilized anti-X F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by LC-MS, SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Each construct can mediate the formation of immunological synapses between T cells and malignant B cells, resulting in T-cell activation. At an E:T ratio of 10:1, using isolated T cells as effector cells, the bsAbs induced potent T-cell-mediated cytotoxicity in various B-cell malignancies, including Burkitt lymphomas (Daudi, Ramos, Namalwa), mantle cell lymphoma (Jeko-1), and acute lymphoblastic leukemia (Nalm-6). A non-tumor binding control, (14)-3s, induced only moderate T-cell killing at >10 nM. The nature of the antigen/epitope, particularly its size and proximity to the cell surface, appears to be more important than antigen density for T-cell retargeting potency (Table 1). It is likely that (20)-3s is consistently more potent than (19)-3s and (C2)-3s, even when the expression of CD19 or HLA-DR is considerably higher than CD20, as seen with Namalwa and Jeko-1, respectively. This is likely because the CD20 epitope comprises a small extracellular loop having close proximity to the cell surface. When compared directly using Daudi, (22)-3s was the least potent. Compared to CD19 and CD20, CD22 is expressed at the lowest density, is a rapidly internalizing antigen, and its epitope is further away from the cell surface; each of these factors may contribute to its reduced potency. Finally, sensitivity to T-cell retargeted killing is cell-line-dependent, as observed using (19)-3s, where Raji (IC50 >3 nM) is largely unresponsive yet Ramos (IC50 = 2 pM) is highly sensitive, even though the former expresses higher CD19 antigen density. Conclusions (19)-3s, (20)-3s, (22)-3s, and (C2)-3s can bind T cells and target B cells simultaneously and induce T-cell-mediated killing in vitro. The modular nature of the DNL method allowed the rapid production of several related conjugates for redirected T-cell killing of various B-cell malignancies, without the need for additional recombinant engineering and protein production. The close proximity of the CD20 extracellular epitope to the cell surface results in the highest potency for (20)-3s, which is an attractive candidate bsAb for use in this platform. We are currently evaluating the in vivo activity of these constructs to determine if this novel bsAb format offers additional advantages. Disclosures: Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other.

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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 ◽

10.1182/blood.v126.23.5118.5118 ◽

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.

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Constitutive Chemokine Receptor Expression in B-Cell Non-Hodgkin’s Lymphoma.

Blood ◽

10.1182/blood.v110.11.3590.3590 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3590-3590

Author(s):

Michelle S. Bryson ◽

Ruth F. Jarrett ◽

Lesley Sheild ◽

Gerard J. Graham

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Hodgkin’S Lymphoma ◽

Memory T Cells ◽

Cell Lymphoma ◽

Hodgkin's Lymphoma ◽

Mantle Cell

Abstract Chemokines are small peptides (∼8-14KDa) that play an essential role in both the innate and adaptive immune system. Chemokines are primarily involved in leukocyte trafficking, but are also involved in a number of cellular mechanisms. They elicit their effect through G-protein coupled receptors, the chemokine receptors (CKR). Functionally chemokines and their receptors are classified as inflammatory or constitutive. Constitutive CKRs and their ligands have a role in numerous diseases including malignancy, chronic inflammation and HIV infection. This study aimed to examine constitutive CKR expression in sub-types of B-cell NHL, of which there are limited studies so far. Lymph node preparations from patients with NHL were examined by flow cytometry using antibodies to CD20, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR4 and CXCR5. The percentage of CD20 positive cells expressing the CKR under investigation was then calculated. The following cases were examined; follicular lymphoma (FL), n=11, Diffuse large B-cell lymphoma (DLBCL), n=11, mantle cell lymphoma (MCL) n=17, Burkitt’s lymphoma (BL), n=9 and MALT lymphoma, n=10. A number of differences between NHL sub-types were detected. FL cases generally had a lower expression of all the CKRs. CXCR5 and CXCR4 expression was high in all sub-types (>84% of B-cells) with no significant differences found, this would be expected as these CKRs are widely expressed in all B-cells. CCR10 expression was low or absent, with no significant differences detected. CCR6 and CCR9 show highest expression in MALT lymphomas, consistent with previous studies, but in comparison with other sub-types the differences was not significant. The most significant results were found with CCR7 and CCR4. CCR7 is expressed on naive T-cells, memory T-cells, B-cells and dendritic cells and is involved in the homing of lymphocytes to lymph nodes. CCR7 is currently the second most commonly reported CKR to be upregulated in malignancy, after CXCR4 and is related. We found very high levels of CCR7 in Mantle cell lymphoma (>90% of B-cells) as compared to other sub-types (p=0.005). CCR4 is expressed on Th2 and Treg lymphocytes, memory T cells and in a small subset of mature B-cells. CCR4 expression in T-cells has been correlated with an adverse prognosis in T-cell NHL and Hodgkin’s lymphoma, yet no systematic studies looking at CCR4 expression in B-cell neoplasms has been reported. These results showed a significant increase in CCR4 expression (>50% of B-cells) in DLBCL, MCL, MALT and BL as compared to FL (p<0.0001). We showed that there are differences in constitutive CKR expression in the different B-cell NHL types, with CCR4 expression being the most interesting finding. How CCR4 expression relates to prognosis in these lymphomas is as yet unknown but is under investigation. Targeting of the chemokine system using anti-CCR4 is already being used in clinical trials for T-cell neoplasms, and may be of potential benefit in selected B-cell neoplasms. Furthermore, the development of anti-CCR7 strategies may prove to be of benefit in the traditionally poor prognosis MCL patients.

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Activity of Bruton's Tyrosine Kinase (Btk) Inhibitor PCI-32765 in Mantle Cell Lymphoma (MCL) Identifies Btk As a Novel Therapeutic Target,

Blood ◽

10.1182/blood.v118.21.3688.3688 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3688-3688 ◽

Cited By ~ 3

Author(s):

Sabine Ponader ◽

Sriram Balasubramanian ◽

Lan V Pham ◽

Jun Chen ◽

Archito T. Tamayo ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Research Funding ◽

Actin Polymerization ◽

Cell Lymphoma ◽

B Cell Malignancies ◽

Mantle Cell ◽

Bcr Signaling ◽

Btk Inhibitor

Abstract Abstract 3688 B cell receptor (BCR) signaling is critically involved in the progression of several B cell malignancies, but its role in mantle cell lymphoma (MCL) remains incompletely defined. Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling and can be selectively and irreversibly inhibited by PCI-32765, which is emerging as a new, molecularly targeted therapy for patients with B cell malignancies. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 & MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75–4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 & Jeko-1 all required >10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at <100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which are surrogate biomarkers for BCR-derived activation of neoplastic B cells (Burger JA et al., Blood 113:3050–8, 2009) with greatest increase in JVM-13 and Rec-1 cells. Pre-treatment of these two MCL lines with PCI-32765 significantly inhibited CCL3 and CCL4 secretion in a dose dependent fashion with total abrogation of chemokine secretion at concentrations of 10 mM PCI-32765 (see Figure). Early clinical data indicate that PCI-32765 induces a rapid reduction in lymphadenopathy accompanied by a transient lymphocytosis (in CLL, but also in MCL patients), presumably due to mobilization of the malignant B cells from the tissue compartments into the peripheral blood. Therefore, we analyzed the effect of PCI-32765 (conc. 0.5 and 1 mM) on MCL responses to a lymph node homing chemokine, CXCL13. We found that CXCL13-induced actin polymerization in Rec-1 cells was significantly reduced by PCI-32765, even at lower concentration. We conclude that MCL cells express functional Btk, which is involved in BCR signaling in MCL cells. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling, chemokine secretion, and interferes with MCL cell actin polymerization. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic. Disclosures: Balasubramanian: Pharmacyclics: Employment. Chen:Pharmacyclics: Employment. Wang:Pharmacyclics: Research Funding. O'Brien:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Burger:Cellgene: Consultancy; Pharmacyclics: Consultancy, Research Funding; Genzyme: Consultancy; Calistoga: Research Funding; Noxxon: Consultancy, Research Funding.

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DUSP1/6 Inhibition Reduces Tumor Cells and Activates Immune Response in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-117052 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2857-2857

Author(s):

Martina Braun ◽

Veronika Ecker ◽

Tanja Neumayer ◽

Markus Muschen ◽

Jürgen Ruland ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Immune Cells ◽

Healthy Donor ◽

Lymphocytic Leukemia ◽

Downstream Signaling ◽

Bcr Signaling

Abstract Chronic lymphocytic leukemia (CLL) is characterized by an expansion of monoclonal, mature B cells that carry auto/polyreactive B cell receptors (BCRs). It is one of the most prevalent B cell malignancies in Western countries. Over the past decade, extensive research has led to significant improvement in understanding its pathogenesis, particularly in regard to the BCR signaling pathway. This has led to the development of novel and promising treatment options, however, none of these approaches are curative and relapses are challenging to treat. Until now allogeneic hematopoietic stem cell transplantation remains the only treatment option for durable remission in poor-risk disease. The therapeutic difficulties result from the heterogeneity, the advanced age of the patients, chemoresistance and relapse of the disease. Here, we test a novel strategy for simultaneously targeting the malignant cells and reverting the CLL-suppressed immune response. Stimulation of the antigen receptors of immune cells readily induces proliferation, differentiation and functional activation. However, strong binding of antigen can also induce negative selection in lymphocytes. CLL B cells constitutively signal through their BCR and thus we set out to investigate whether they rely on the counterbalancing, negative regulation of specific downstream signaling pathways, in particular the mitogen-activated protein kinase (MAPK) pathway. The dual specificity phosphatases 1 and 6 (DUSP1 and DUSP6) dephosphorylate extracellular-signal regulated kinase 1/2 (ERK1/2) and thereby limit ERK1/2 activation. These molecules are frequently downregulated in solid tumors (Khor et al., Int. J. Med.Sci 2013; Okudela et al., Am. J. Pathol. 2009). We therefore set out to analyze the expression levels of DUSP1 and DUSP6 in CLL and found them readily expressed at various levels, comparable to normal B cells. To determine the functional relevance of DUSP1 and DUSP6, we blocked their phosphatase function using the small molecule inhibitor BCI. By treatment with BCI, we induced hyperactivation of the MAPK signaling cascade followed by cell death of the CLL cells. Interestingly, the induction of cell death is specific for CLL cells and does not occur to the same extent in other malignant B cell lymphoma cells or healthy donor-derived B cells. This deleterious effect of BCI was evident in primary patient-derived CLL cells as well as in the CLL-like cell lines MEC-1 and EHEB and in CLL cells derived from the T cell leukemia/lymphoma 1 (TCL1)-driven mouse model. To further investigate the downstream signaling event upon BCI treatment, we conducted a global phosphoproteome analysis. After treatment of primary CLL cells with BCI, the most significant alterations were within the BCR signaling pathway, including hyperphosphorylation of ERK1/2 and followed by a rapid induction of a DNA damage response. These results were validated by immunoblot analysis of human and murine CLL cells and were not detected in BCI-resistant cell lines. Beside the direct effects of BCI on the CLL cells we set out to investigate effects on other immune cells, directly by BCI and secondary via CLL cells treated with BCI. Indeed, we observed changes in immune cell compartment: BCI-treated CLL patient-derived peripheral blood mononuclear cells (PBMCs) resulted in selective enrichment of cytotoxic T cells. Furthermore, BCI treatment of CLL cells fed with Ovalbumin, in co-culture with or without BMDCs and OT-I cytotoxic CD8 T cells (specifically recognizing the SINFEKL peptide), resulted in the induction of immunogenic cell death of CLL cells. This was evidenced by enhanced antigen-specific T cell proliferation and release of the high mobility group box 1 protein (HMGB1). To investigate indirect effects of BCI, we treated CLL cells with BCI at sublethal doses, then washed the cells and co-cultured these primary CLL cells or CLL cell lines with healthy donor-derived PBMCs. The frequencies of CLL-induced myeloid-derived suppressor cells (MDSCs) as well as regulatory T cells (Tregs) were reduced after co-cultivation of PBMCs with BCI-pretreated CLL cells. Taken together, our data indicate that negative feedback inhibition reduces CLL content by induction of immunogenic cell death and activates immune cells to target the CLL-induced dysfunction of the immune system. We therefore propose that inhibition of DUSP1/6 is a promising therapeutic approach for CLL. Disclosures No relevant conflicts of interest to declare.

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Autonomous Ca2+ Oscillations Reflect Oncogenic BCR-Signaling in Multiple B-Cell Malignancies and Are Essential for Survival and Proliferation

Blood ◽

10.1182/blood-2018-99-117315 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1373-1373

Author(s):

Kohei Kume ◽

Liting Chen ◽

Jae-Woong Lee ◽

Markus Muschen

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Oncology Group ◽

B Cell Malignancies ◽

Myeloma Cells ◽

Expression Levels ◽

Mantle Cell ◽

Bcr Signaling ◽

Median Expression

Abstract Background: Nuclear factor of activated T cells (NFAT) factors regulate activation and Ca2+ signaling in B-cells. Store-operated Ca2+ entry (SOCE) is regulated by Orai1 and Stim1 and upstream of NFAT. We previously reported on the observation of autonomous oscillatory Ca2+ signal activity in BCR-ABL1-driven B-ALL. Autonomous Ca2+ oscillations may provide oncogenic survival signals to B-ALL cells, however their significance and mechanism remained unclear. Results: Here we found that autonomous Ca2+ oscillations are common across multiple subtypes of B-cell lineage ALL and mature B-cell lymphoma and reflect downstream survival and proliferation signals from oncogenic BCR-signaling or oncogenes that mimic active BCR-signaling (e.g. ABL1-kinase fusions, CD79B mutation, EBV-encoded oncoprotein LMP2A, RAS-pathway lesions). By contrast, multiple myeloma and Hodgkin's lymphoma that lack BCR-expression and function also lack Ca2+ oscillations (Figure, top panel). As Orai1 and Stim1/2 are essential SOCE-effector genes, we performed genetic experiments to test the impact of Cre-mediated deletion of Orai1 and Stim1/2 in BCR-ABL1 and NRASG12D-dependent models of B-ALL. Inducible deletion of Orai1 or Stim1/2 not only abrogated SOCE but also autonomous Ca2+ oscillations. Signal amplitudes of residual autonomous Ca2+ oscillations were significantly reduced (P < 0.0001; Figure, bottom panel). Further, deletion of either Orai1 or Stim1/2 induced cell death and abrogated colony-forming capacity in both models of B-ALL. In agreement with these findings, NFATc1 no longer translocated to the nucleus upon Cre-mediated ablation of Orai1 or Stim1/2 induced, which reflected functional inactivation of NFATc1. These results demonstrated that Orai1- and Stim1/2-mediated SOCE signaling and autonomous Ca2+ oscillations are critical in BCR-ABL1 and NRASG12D-dependent B-ALL. A NFAT-calcineurin association inhibitor, INCA-6, was tested for its ability to suppress NFATc1 and autonomous Ca2+ signaling in patient-derived xenograft (PDX) models of B-ALL, mantle cell lymphoma and DLBCL. Treatment with INCA-6 suppressed survival and proliferation signals in all six PDX of B-ALL, mantle cell lymphoma and DLBCL but not multiple myeloma cells. Unlike myeloma cells, B-ALL, mantle cell and DLBCL cells expressed a functional (pre-)BCR. These findings suggest that the SOCE-NFAT pathway is linked to Ca2+ signaling downstream of a functional BCR- or oncogenic BCR-mimics. Furthermore, to determine whether high expression levels of ORAI1, STIM1, STIM2 and NFATC1 represents a biomarker of clinical outcome for patients with B-ALL, we segregated patients from two clinical trials (Children's Oncology Group P9906 (n=207) and Eastern Cooperative Oncology Group (E2993; n=215)) into two groups on the basis of higher or lower than median expression levels of ORAI1, STIM1, STIM2 and NFATC1 at the time of diagnosis. Higher than median expression levels of each of these four genes at the time of diagnosis predicted shorter overall and relapse-free survival (P < 0.02 or lower for each of these genes). These findings identify SOCE-NFAT signal as a novel biomarker with potential use in risk stratification of children and adults with B-ALL. Conclusions: We identified Orai1 and Stim1 as central mediators of SOCE. Most BCR-dependent B-cell malignancies are driven by oncogenic BCR-signals, which result in autonomous Ca2+ oscillations. While the significance of autonomous Ca2+-oscillations remains unclear, deletion of Orai1 and Stim1/2 resulted in a complete loss of Ca2+ oscillations, loss of NFATc1-activation and cell death. We conclude that previously unrecognized Ca2+-oscillations downstream of oncogenic BCR-signaling are required for survival and proliferation of B-ALL and B-cell lymphoma cells. Pharmacological inhibition of SOCE (Orai1 and Stim1/2) or NFATC1 (e.g. INCA-6) represents a selective strategy to disrupt autonomous Ca2+ oscillations and oncogenic BCR-signaling in a broad range of B-cell malignancies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Targeted Activation of B Cell Autoimmunity Checkpoints in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3716.3716 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3716-3716

Author(s):

Zhengshan Chen ◽

Huimin Geng ◽

Clifford A. Lowell ◽

Arthur Weiss ◽

Stephen P. Hunger ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Negative Selection ◽

Lymphoblastic Leukemia ◽

Cell Selection ◽

Surface Receptors ◽

Bcr Signaling

Abstract Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in "death by neglect". If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, Ph+ ALL cells lack expression of pre-BCR signaling chains Iga and Igb, indicating lack of a functional pre-BCR. Reconstitution of Iga and Igb was indeed sufficient to induce cell death in BCR-ABL1 ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. These findings suggest that Ph+ ALL are uniquely sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph+ ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph+ ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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