Daratumumab, a Human CD38 Antibody Induces Apoptosis of Myeloma Tumor Cells Via Fc Receptor-Mediated Crosslinking.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2974-2974 ◽  
Author(s):  
J. H. Marco Jansen ◽  
Peter Boross ◽  
Marije B. Overdijk ◽  
Jeroen J. Lammerts van Bueren ◽  
Paul W.H.I. Parren ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Cell Line ◽  
Tumor Cells ◽  
Mechanism Of Action ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Killing Activity

Abstract Abstract 2974 Daratumumab (DARA) is a human CD38 monoclonal antibody with broad-spectrum killing activity. DARA is in clinical development for multiple myeloma (MM) and has potential in other hematological tumors on which CD38 is expressed. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. The killing activity of DARA on CD38-expressing tumor cells has previously been mainly attributed to complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In this study we explored whether DARA could also kill CD38-positive cells via induction of apoptosis after immobilization of DARA via Fc receptors (FcRs) in vitro. To this end, target cells were co-incubated in vitro with hFcgRI-expressing cells, lacking ADCC activity, in the absence or presence of DARA. Apoptosis was measured in flow cytometry for annexin-V/7AAD positivity. Treatment of the Burkitt's lymphoma cell line Ramos with DARA in combination with hFcgRI-expressing cells, led to a significant enhancement of annexin–V/7AAD positive cells (p<0.05 Dunn's multiple comparison test). Similar results were obtained with patient-derived multiple myeloma cell lines L363 and UM9, which are transduced with CD38 to obtain comparable expression levels to primary myeloma cells. This indicates that FcR-mediated crosslinking of DARA induces apoptosis which might contribute to the mechanism of action. We are currently performing experiments to investigate the contribution of DARA-mediated apoptosis in vivo in tumor growth inhibtion. Therefore, DARA efficacy is studied in a peritoneal syngeneic mouse model with EL4-CD38 cells, a mouse lymphoma cell line transfected with human CD38. The EL4-CD38 cells showed efficient apoptosis induction after FcR-mediated crosslinking of DARA in vitro. For this mouse model we will make use of NOTAM mice, which have a functionally inactive FcR associated gamma chain leading to cell membrane expression of all FcRs, but without signaling capacity (de Haij et al. Cancer Research 2010). Leucocytes of these mice are capable of antibody crosslinking via FcRs, but incapable of inducing cytotoxicity via ADCC. In conclusion, in vitro data suggest that FcR-mediated crosslinking of DARA induces apoptosis of CD38 expressing tumor cells, and can thus be considered an additional mechanism of action for DARA. Disclosures: Jansen: Genmab BV: Research Funding. Overdijk:Genmab BV: Employment. Lammerts van Bueren:Genmab BV: Employment. Parren:Genmab BV: Employment.

Inhibition Effects of baicalin on Lymphoma Cell Line CA46 in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5053-5053
Author(s):  
Jian Da Hu ◽  
Yi Huang ◽  
Yingyu Chen ◽  
Tiannan Wei ◽  
Tingbo Liu ◽  
...  
Keyword(s):  
Cell Line ◽  
Biological Effects ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Control Group ◽  
Dependent Manner ◽  
Lymphoma Cell Line ◽  
Proliferation Inhibition

Abstract Baicalin is a traditional Chinese medicine with multiple biological effects. Some researches showed baicalin has anti-tumor effects in solid tumor, such as prostate cancer. In order to investigate its effects on proliferation inhibition and apoptosis induction in human lymphoma cell, we treated Burkitt lymphoma cell line CA46 with baicalin in vitro and in vivo of CA46 xenograft. Baicalin remarkably inhibited the cell proliferation, with an IC50 value of 10μM. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, which was detected by Annexin V FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cell decreased in a time-dependent manner. Western-Blot showed that the protein expressions of c-myc, bcl-2, procaspase-3 and PARP(116KD) in baicalin treated CA46 cell were down-regulated, while the expression of PARP(85KD) increased. Based on the results in vitro, we investigated in vivo efficacy of baicalin, alone or in combination with cytotoxic drug VP16, for treatment in CA46 nude mice xenograft. Baicalin with the dosage of 40mg/kg/d and 80kg/mg/d could remarkably inhibit the growth of the tumor compared with control group. Combination of baicalin and VP16 had better anti-tumor effects. Histological examination of tumor samples showed more necrotic cells in treated groups. And obvious apoptosis could be observed by electron microscope. No adverse events were found in treated groups. From above we could conclude that baicalin could efficiently induce proliferation inhibition and apoptosis of CA46 cells in vitro and in vivo, which may be related with the down-regulation of c-myc and bcl-2 expressions, as well as the up-regulation of caspase-3 activity.

scholarly journals CD38 x ICAM1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2413-2413
Author(s):  
Xiaocheng Chen ◽  
Oi Kwan Wong ◽  
Leonard Post
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Bispecific Antibody ◽  
Lymphoma Cell ◽  
Cd38 Expression ◽  
Novel Approach

Abstract Targeting cluster of differentiation 38 (CD38) with monoclonal antibodies has resulted in outstanding responses in patients with multiple myeloma (MM). However, a significant portion of patients failed to respond and nearly all patients eventually relapsed. Furthermore, daratumumab, an anti-CD38 antibody, only showed limited monotherapy activity in relapsed/refractory Non-Hodgkin lymphoma (NHL) patients in a phase 2 study. One of the mechanisms of resistance has been partially attributed to lower CD38 expression. Intercellular adhesion molecule 1 (ICAM1), an immunoglobulin (Ig)-like cell adhesion molecule, is highly expressed in multiple myeloma and lymphoma. Antibody against ICAM1, bersanlimab (BI505, BioInvent), was well-tolerated, but only showed limited clinical efficacy in MM patients. Here, we generated bispecific CD38 x ICAM1 antibody to target ICAM1 + tumor types with low to medium CD38 expression. RNA sequencing (RNAseq) results from the Cancer Cell Line Encyclopedia (CCLE) database showed that ICAM1 is highly expressed on myeloma and lymphoma cell lines. ICAM1 expression levels for selected myeloma and lymphoma cell lines were then validated using flow cytometry. The CD38 x ICAM1 bispecific antibody was constructed by paring a novel CD38 antibody and a novel ICAM1 antibody through an asymmetric three chain knob-into-hole format. The bispecific antibody showed potent in vitro antibody-dependent cellular cytotoxicity (ADCC) activities on ICAM1 + tumor cells with medium to low CD38 levels, where daratumumab has low or minimal effect. The bispecific antibody also showed potent in vitro antibody-dependent cellular phagocytosis (ADCP) activities on cell lines with a range of CD38 expression. The CD38 x ICAM1 bispecific antibody further demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma cell line-derived xenograft (CDX) models, including cell lines with low to medium CD38 expression. We then evaluated CD38 and ICAM1 expressions in lymphoma patient-derived xenograft (PDX) samples by RNAseq. Among the 37 PDX samples, 27 of them showed ICAM1 expression above 2 5 fragments per kilobase of transcript per million map reads (FPKM). On the contrary, there is a wide range of CD38 expression levels with only 6 samples having CD38 expression above 2 5 FPKM. The ICAM1 and CD38 expressions in the selected PDX samples were further validated with IHC staining. Most importantly, the CD38 x ICAM1 bispecific antibody showed complete tumor inhibition in a rituximab-resistant lymphoma PDX model, whereas daratumumab only showed minimal efficacy. In conclusion, the CD38 x ICAM1 bispecific antibody demonstrated improved efficacy and specificity toward CD38 + and ICAM1 + tumor cells and represents a novel approach for treating multiple myeloma and lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals In vivo growth of a murine lymphoma cell line alters regulation of expression of HSP72.

1995 ◽  
Vol 15 (2) ◽  
pp. 1071-1078 ◽  
Author(s):  
S Davidson ◽  
P Høj ◽  
T Gabriele ◽  
R L Anderson
Keyword(s):  
Heat Shock ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Regulation Of Expression ◽  
Heat Shock Element ◽  
Stressed Cells

We have identified a murine B-cell lymphoma cell line, CH1, that has a much-diminished capacity to express increased levels of heat shock proteins in response to heat stress in vitro. In particular, these cells cannot synthesize the inducible 72-kDa heat shock protein (HSP72) which is normally expressed at high levels in stressed cells. We show here that CH1 fails to transcribe HSP72 mRNA after heat shock, even though the heat shock transcription factor, HSF, is activated correctly. After heat shock, HSF from CH1 is found in the nucleus and is phosphorylated, trimerized, and capable of binding the heat shock element. We propose that additional signals which CH1 cells are unable to transduce are normally required to activate hsp72 transcription in vitro. Surprisingly, we have found that when the CH1 cells are heated in situ in a mouse, they show normal expression of HSP72 mRNA and protein. Therefore, CH1 cells have a functional hsp72 gene which can be transcribed and translated when the cells are in an appropriate environment. A diffusible factor present in ascites fluid is capable of restoring normal HSP72 induction in CH1 cells. We conclude that as-yet-undefined factors are required for regulation of the hsp72 gene or, alternatively, that heat shock in vivo causes activation of hsp70 through a novel pathway which the defect in CH1 has exposed and which is distinct from that operating in vitro. This unique system offers an opportunity to study a physiologically relevant pathway of heat shock induction and to biochemically define effectors involved in the mammalian stress response.

scholarly journals The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.

1992 ◽  
Vol 118 (6) ◽  
pp. 1443-1453 ◽  
Author(s):  
J Jongstra-Bilen ◽  
P A Janmey ◽  
J H Hartwig ◽  
S Galea ◽  
J Jongstra
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Fusion Proteins ◽  
Specific Protein ◽  
Lymphoma Cell ◽  
Actin Binding ◽  
Lymphoma Cell Line ◽  
Basic Domain ◽  
Acidic Domain

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.

A Novel Celecoxib Derivative, OSU03012, Induces Cytotoxicity in Primary CLL Cells and Transformed B-Cell Lymphoma Via a Caspase and Bcl-2 Independent Mechanism.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3471-3471
Author(s):  
Amy Johnson ◽  
Lisa Smith ◽  
Jiuxiang Zhu ◽  
Nyla Heerema ◽  
Sara Guster ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cell Line ◽  
Mechanism Of Action ◽  
Caspase 3 ◽  
Annexin V ◽  
In Vitro Activity ◽  
Caspase 9 ◽  
Over Expression

Abstract Chronic lymphocytic leukemia (CLL) is an incurable adult leukemia characterized by disrupted apoptosis. While the majority of patients with CLL are asymptomatic at diagnosis, most progress and require therapy. Identification of new targets and therapeutic agents is therefore a high priority for the treatment of CLL. Synthetic chemistry yielded derivatives of the COX-2 inhibitor, celecoxib, with increased ability to induce apoptosis in the 1–10 μ M range in prostate cancer cells, a similar proposed mechanism of action, and increased in vivo activity in a murine prostate cancer xenograft model. Based upon these data, a Rapid Access to Intervention Development (RAID) proposal is underway to generate OSU03012 for clinical studies in prostate cancer. In addition, we are examining the biologic effects of these new agents in primary CLL cells and lymphoblastic cell lines, showing a novel mechanism of cell killing independent of caspase activation and bcl-2 over-expression. To determine the in vitro activity against CLL cells, 11 CLL patient PBMCs were incubated in various concentrations of OSU03012. The LC50 at 24 hrs was 7.12μM and decreased to 5.45μM at 72 hrs. We show both early (annexin-V positive) and late (both annexin-V/PI positive) apoptosis concurrent with loss of mitochondrial membrane potential typical of apoptosis. These data suggest OSU03012 is highly cytotoxic toward CLL cells in vitro at doses well below those attainable without toxicity in a murine model. Additionally, we show that OSU03012 mediates apoptosis by activation of the intrinsic, mitochondrial pathway of apoptosis but also activates alternative caspase independent cell death pathways. CLL cells from 8 patients were incubated in 10μM OSU03012 for 24 hrs and assessed for caspase-3 and PARP. Immunoblots reveal a dose dependent increase in active caspase-3 concurrent with a decrease in the pro-form. This occurred concurrently with the appearance of the 85 kD cleaved product of PARP that is a known downstream target of caspase-3. In the same 8 patient lysates we saw no change in the inactive pro-form of caspase-8, but consistent processing of caspase-9. These data suggest that OSU03012 in part utilizes the intrinsic pathway of apoptosis to promote CLL cell death. Incubation of CLL cells with z-VAD-fmk and OSU03012 did not abrogate cell death, but eliminated processing of caspase-9, caspase-3 and PARP, suggesting that this agent also activates caspase independent mechanisms of cell death. Given the caspase dependent and independent pathways utilized by OSU03012, we assessed the dependence of cell death on bcl-2 expression. Here we show that bcl-2 over-expression in the 697 lymphoblastic cell line greatly diminishes the apoptosis observed with fludarabine, but potent apoptosis is equally observed with OSU03012 compared to the empty vector cell line. Furthermore, in the bcl-2 over-expressing cell line, caspase-3 and PARP cleavage was not observed despite equivalent apoptosis supporting further multiple mechanisms of cell killing induced by OSU03012. In summary, OSU03012 is an oral bioavailable therapeutic agent that has potent in vitro activity against primary CLL cells. This cytotoxicity is mediated by both caspase dependent and independent pathways and can overcome bcl-2 over-expression. These data provide support for further investigation of the mechanism of action of OSU03012 in CLL cells and performance of early Phase I studies in CLL as part of the RAID process.

scholarly journals Preclinical Validation of Ubiquitin Receptor PSMD4 As Therapeutic Target in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4403-4403
Author(s):  
Yan Song ◽  
Ting DU ◽  
Arghya Ray ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Line ◽  
Therapeutic Potential ◽  
Statistical Significance ◽  
Annexin V ◽  
Endoplasmic Reticulum Stress Response ◽  
In Vivo Models

Background and Rationale Our preclinical studies have focused on Identification and validation of components within the ubiquitin proteasome system which can be targeted with novel therapies to overcome proteasome-inhibitor (PI)-resistance in multiple myeloma (MM). Our siRNA screening studies identified ubiquitin Receptor (UbR) PSMD4/Rpn10 as a mediator of MM cell growth and survival. Rpn10 is localized on the 19S regulatory lid of the proteasome, and plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. Here, we show that inhibition of Rpn10 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. Materials and Methods Rpn10 knockout 293 cell line was generated using CRISPR/Cas9. Rpn10-inducible knockdown (KD) MM.1S cell line was generated using shRNA. Drug sensitivity, cell viability, and apoptosis assays were performed using WST/CellTiter-Glo assay, and Annexin V staining, respectively. Cell signaling and caspase activity were determined by western blotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2012, 22:345-358). A xenograft human MM model was used to characterize the role of Rpn10 on tumor progression. Statistical significance was assessed with Student's t test. Results 1) Analysis of MM patient gene expression profiling database showed that Rpn10 expression inversely correlates with overall survival (n=175; p= 0.00064). 2)Real-time PCR, immunoblotting, and immunohistochemistry of MM patient bone marrow showed higher Rpn10 levels in patient MM cells and MM cell lines versus normal cells. 3)Transient transfection of MM cells with Rpn10-siRNA decreased their viability; conversely, transfection with Rpn10-WT specifically rescued cells from growth-inhibitory activity of Rpn10-siRNA. Immunoblot analysis confirmed significant knockdown of Rpn10 by Rpn10-siRNA versus scrambled (scr)-siRNA, and restoration of Rpn10 levels in cells transfected with Rpn10-WT versus Rpn10-siRNA. 4)Genetic blockade of Rpn10 decreased viability in bortezomib-resistant MM cells. 5)CRISPR/Cas9-mediated knockout (KO) of Rpn10 decreased cell growth. 5)Both Rpn10-KO and inducible Rpn10-KD cells showed elevated levels of polyubiquitylated proteins. 6)Inhibition of Rpn10 induced apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling. 7)Reduced tumor growth was noted in mice xenografted with Rpn10-KD MM.1S cells versus mice engrafted with WT-MM.1S cells. Conclusion Our data demonstrate the therapeutic potential of targeting ubiquitin receptor Rpn10/PSMD4, and provide the preclinical basis for development of Rpn10 inhibitors to overcome PI-resistance in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

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