Purpose To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ CLL subtype. Experimental design The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP) or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models. Results Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo, while exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by down-regulation of the matrix metalloproteinase MMP9. Conclusions These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL.

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.

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Purpose: To uncover the signaling pathways underlying follicular lymphoma-follicular dendritic cells (FL-FDC) cross-talk and its validation as new targets for therapy.Experimental Design: FL primary cells and cell lines were cocultured in the presence or absence of FDC. After 24 and 48 hours, RNA was isolated from FL cells and subjected to gene expression profiling (GEP) and data meta-analysis using DAVID and GSEA softwares. Blockade of PI3K pathway by the pan-PI3K inhibitor BKM120 (buparlisib; Novartis Pharmaceutical Corporation) and the effect of PI3K inhibition on FL-FDC cross-talk were analyzed by means of ELISA, RT-PCR, human umbilical vein endothelial cell tube formation, adhesion and migration assays, Western blot, and in vivo studies in mouse FL xenografts.Results: GEP of FL-FDC cocultures yields a marked modulation of FL transcriptome by FDC. Pathway assignment by DAVID and GSEA software uncovered an overrepresentation of genes related to angiogenesis, cell adhesion, migration, and serum-response factors. We demonstrate that the addition of the pan-PI3K inhibitor BKM120 to the cocultures was able to downregulate the expression and secretion of proangiogenic factors derived from FL-FDC cocultures, reducing in vitro and in vivo angiogenesis. Moreover, BKM120 efficiently counteracts FDC-mediated cell adhesion and impedes signaling and migration induced by the chemokine CXCL12. BKM120 inhibits both constitutive PI3K/AKT pathway and FDC-or CXCL12-induced PI3K/AKT pathway, hampers FDC survival signaling, and reduces cell proliferation of FL cells in vitro and in mouse xenografts.Conclusions: These data support the use of BKM120 in FL therapy to counteract microenvironmentrelated survival signaling in FL cells. Clin Cancer Res; 20(13); 3458-71. Ó2014 AACR.

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The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC‐292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC‐292 potently inhibited B‐cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC‐292 reduced tumor load and normalized tumor‐associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC‐292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC‐292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

3935 Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the present study, we have analyzed the potential of targeting CD38 using DARA in two types of B-cell non-Hodgkin lymphoma (B-NHL) (follicular lymphoma (FL) and mantle cell lymphoma (MCL)), and in chronic lymphocytic leukemia (CLL). Flow cytometry analysis demonstrated that MCL and CLL tumor cells show heterogeneous expression of CD38, while FL cells showed invariable high CD38 levels. CD38 has attracted special attention in CLL where high CD38 expression is a marker of bad prognosis (Hamblin et al, Blood 1999 and 2002) and is expressed preferentially in the proliferating fraction of the tumor (Damle RN, Blood 2007). In addition, we have recently shown that high CD38 expression in MCL was associated with resistance to the proteasome inhibitor bortezomib (Pérez-Galán P, Blood 2011). Here, we tested the cytotoxic activity of DARA in tumor cell lines and in fresh tumor cells obtained from patients. DARA did not induce CDC in MCL cell lines (MINO, REC, HBL2, JEKO), irrespective of CD38 expression levels. Also, FL cell lines (WSU-FSCCL, RL) expressing relatively high CD38 levels were insensitive to DARA-induced CDC. This low CDC was associated with high expression of the complement inhibitors CD55 and CD59. In addition, the number of CD38 molecules per cell in these MCL and FL cell lines was lower than that found on the CDC-sensitive Daudi Burkitt lymphoma cell line, suggesting a threshold for CD38-targeted CDC lysis. In the presence of PBMC effector cells obtained from healthy donors, DARA showed significant levels of ADCC in cells from MCL, FL and CLL. In CLL primary cases (n=8) tested, DARA (14–43% lysis) was generally superior or at least equally effective (mean+/−SD=28,78 +/− 9,78) in inducing ADCC as compared to the anti-CD20 antibodies ofatumumab (mean+/−SD=21,35 +/− 15,71) and rituximab (mean+/−SD=29,30 +/− 15,90). The immunomodulatory agent lenalidomide shows considerable single agent activity in MCL, FL and CLL. Interestingly, it has been shown that lenalidomide may be able to increase ADCC, probably via activation of NK cells. We therefore tested whether the combination of lenalidomide and DARA could enhance ADCC. Noteworthy, DARA-induced ADCC in MCL, FL cell lines and primary CLL cells was significantly (p<0,05) enhanced when effector cells were pretreated with the immunomodulatory agent lenalidomide (3 μM, 72 h) with DARA doses ranging from 0,01–1 μg/ml. Finally, CD38 is important for cell migration and adhesion, especially for CXCR4-CXCL12-induced migration of tumor cells (Vaisitti et al, Leukemia 2010). Our preliminary results suggest that in the CLL subtype with high CD38 and more migratory capacity DARA (10–30 μg/ml) inhibits CXCL12/SDF1α mediated migration up to 70%. These results are of high importance because inhibition of tumor cell trafficking to tissue sites as bone marrow and lymph nodes, may be clinically relevant, as increasing evidence indicates that tumor–microenvironment interactions may play an important role in drug resistance and contribute to clinical failures. We are currently validating this in vitro results in a CLL mouse model. Taken together, these results suggest that DARA may be a promising therapeutic agent both for MCL, FL and CLL, in which DARA exerts its effects mainly via ADCC and for CLL also via inhibition of migration of tumor cells. Interestingly, the cytotoxic activity of DARA by ADCC could be further augmented by addition of lenalidomide in these three models. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Perez-Galan:Genmab: Research Funding.

Daratumumab (DARA) is a anti-human CD38 antibody with Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (mΦ), both murine and human in multiple myeloma (MM) and Burkitt lymphoma cells. DARA is currently being evaluated in phase III clinical trials in patients with MM. We have previously reported that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Chronic Lymphoctic Leukemia (CLL), and significantly prolongs overall survival of animals in a systemic CLL mouse model. Here, we present additional data on in vivo mechanism of DARA and its effect on tumor-microenvironment interactions in CLL. We first evaluated whether ADCP contributes to DARA activity both in vitro and in vivo. For in vitro ADCP, mΦ were generated from monocytes of normal PBMCs and stimulated with GM-CSF (10ng/mL, 7 days). CLL cell lines and primary cells were labeled with calcein and incubated for 4h with mΦ at an effector:target ratio of 2:1 in the presence of a fixed mAb concentration of 1 μg/mL, followed by flow cytometric analysis. The amount of remaining CLL target cells (CD19+, CD11b-) was reduced by 3-16%. ADCP defined as percentage of mφ which had phagocytosed, referred to as double positive mΦ (CD11b+, calcein+, CD19-), ranged from 3-10%. To analyze ADCP in vivo, SCID beige mice, devoid of NK cells but with active macrophages, were inoculated intraperitoneally with CLL cells (20×106) and simultaneously treated with a single dose of DARA or isotype control (20mg/kg, n=3-5 per group). Forty-eight hours later, CLL cells were recovered from the intraperitoneal cavity and counted in a flow cytometer (identified as human CD45+/CD19+/CD5+cells). In DARA-treated mice the number of CLL cells recovered was reduced by 42% (n=2, p<0.05) compared to the isotype control group. Remarkably, the decrease in cell number was already detectable 2h after DARA administration. CLL pathogenesis relies on supportive tumor-microenvironment interactions both in the bone marrow (BM) and in the lymph node (LN), and CD38 constitutes a molecular hub integrating proliferative and migratory signals for CLL (Malavasi, F. Blood 2011). We evaluated the effect of DARA on migration and adhesion. In in vitro migrations assays, we have demonstrated that DARA (10-30 μg/mL) inhibited CXCL12/SDF1α-mediated migration up to 70% (n=5). In addition, DARA reduced up to 55% (n=2) of downstream pERK activation, that peaked after 5min of CXCL12/SDF1α stimulation. We analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to BM and spleen in vivo, using NOD/SCID/gamma (NSG) null mice (lacking NK cells and effective macrophages). In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, prior to injection of fresh primary CLL cells (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for human CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05). In addition to migration, CD38 also plays a key role in cell adhesion through interaction with integrins (CD49d/CD29) and with extracellular matrix proteins. We analyzed the effect of DARA on the adhesion of CLL cells to the extracellular matrix vascular-cell adhesion molecule-1 (VCAM-1) mediated by CD49d/CD29. DARA reduced adhesion of CLL cells (n=4), to VCAM-1 by 46±13% (range 27-57) compared to isotype control. By RT-PCR we observed an up-regulation of MMP9 transcripts (average 2 fold, n=2), and DARA abrogated both constitutive MMP9 expression (90% reduction) and VCAM-derived (94% reduction) MMP9 expression. In summary, DARA shows a positive effect on ADCP-mediated anti-tumor activity on CLL cells both in vitro and in vivo. In addition DARA exhibits a strong effect on CLL cell migration and adhesion. Based on these data, we hypothesize that DARA may exert unique and substantial effects on CLL tumor cell growth and contributes to potent therapeutic efficacy in a clinical setting. Disclosures Doshi: Janssen R&D: Employment. Parren:Genmab: Employment, Equity Ownership. Lammerts van Bueren:Genmab : Employment. Pérez-Galán:Genmab: Research Funding.