scholarly journals Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1482-1489 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
JW Fay ◽  
MJ Stone
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Colony Formation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Marrow Cells

Abstract The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.

scholarly journals Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1482-1489
Author(s):  
AW Tong ◽  
JC Lee ◽  
JW Fay ◽  
MJ Stone
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Colony Formation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Marrow Cells

The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.

scholarly journals A Novel Anti-a Proliferation-Inducing Ligand Hapril.01A Monoclonal Antibody Targets Multiple Myeloma Cells in the Bone Marrow Microenvironment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2098-2098
Author(s):  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
Gang An ◽  
Mike Y Zhong ◽  
Michele Cea ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Dna Binding ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cell ◽  
Molecular Mechanisms ◽  
Myeloma Cells ◽  
Micromolar Range

Abstract A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. Unlike BAFF, APRIL is dispensable in B cell homeostasis but more critical in plasma cell differentiation and survival. It has higher affinity than BAFF (nanomolar vs micromolar range) to B cell maturation antigen (BCMA) (nanomolar vs micromolar range) which expresses at high levels in all patient MM cells. APRIL also binds to a common plasma cell (PC) marker syndecan-1 (CD138) to induce signaling cascade via TACI, the other APRIL receptor in PC. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro osteoclast and macrophage cultures were performed by stimulating CD14+ monocytes from MM patient samples with M-CSF/RANKL and M-CSF, respectively. Osteoclasts and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells (BMSC), as confirmed by ELISA and qRT-PCR. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation consistently activated NF-kB, PI3K/AKT, and ERK1/2 signaling in MM cells. Importantly, NF-kB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute and sustain to 4h in all MM cell lines after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with significantly decreased NF-kB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted viability and colony formation of MM cells, in the presence of osteoclasts or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES and MDC (ccl22), were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Disclosures van Eenennaam: BioNovion: Employment. Elsas:BioNovion: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
G. Teoh ◽  
Y.-T. Tai ◽  
M. Urashima ◽  
S. Shirahama ◽  
M. Matsuzaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
Multiple Myeloma Cell ◽  
Cd40 Activation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

scholarly journals Immunotherapy of Multiple Myeloma With a Monoclonal Antibody Directed Against a Plasma Cell-Specific Antigen, HM1.24

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3179-3186 ◽  
Author(s):  
Shuji Ozaki ◽  
Masaaki Kosaka ◽  
Shingo Wakatsuki ◽  
Masahiro Abe ◽  
Yasuo Koishihara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Plasma Cell ◽  
Severe Combined Immunodeficiency ◽  
Specific Antigen ◽  
Chemotherapeutic Agents ◽  
Scid Mice ◽  
Dependent Manner ◽  
Rpmi 8226

Abstract Multiple myeloma remains an incurable malignancy because of marked resistance of tumor cells to conventional chemotherapeutic agents. Alternative strategies are needed to solve these problems. To develop a new strategy, we have generated a monoclonal antibody (MoAb), which detects a human plasma cell-specific antigen, HM1.24. In this report, we evaluated the in vivo antitumor effect of unconjugated anti-HM1.24 MoAb on human myeloma xenografts implanted into severe combined immunodeficiency (SCID) mice. Two models of disseminated or localized tumors were established in SCID mice by either intravenous or subcutaneous injection of human myeloma cell lines, ARH-77 and RPMI 8226. When mice were treated with a single intraperitoneal injection of anti-HM1.24 MoAb 1 day after tumor inoculation, the development of disseminated myeloma was completely inhibited. In mice bearing advanced tumors, multiple injections of anti-HM1.24 MoAb reduced the tumor size and significantly prolonged survival, including tumor cure, in a dose-dependent manner. The proliferation of cultured human myeloma cells was inhibited in vitro by anti-HM1.24 IgG-mediated complement-dependent cytotoxicity, but not by the antibody alone. Moreover, spleen cells from SCID mice mediated antibody-dependent cell cytotoxicity against RPMI 8226 cells. These results indicate that anti-HM1.24 MoAb can be used for immunotherapy of multiple myeloma and related plasma cell dyscrasias.

The sitotoxic effect of gemcitabine on multiple myeloma (RPMI-8226) and Ig G plasma cell leukemia (ARH 77) cell lines

2012 ◽  
Vol 54 (4) ◽  
pp. 263 ◽  
Author(s):  
Zehra Coban ◽  
Ferit Avcu ◽  
Ali Ural ◽  
Okan Kuzhan ◽  
Sefik Guran
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cell Leukemia ◽  
Cell Leukemia ◽  
Rpmi 8226 ◽  
Ig G

C-met Receptor as a Potential Target for the Treatment of Patients with Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3395-3395
Author(s):  
Marcin Majka ◽  
Artur Jurczyszyn ◽  
Anna Zebzda ◽  
Wojciech Czogala ◽  
Ewa Lesko ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Protein Level ◽  
Bone Marrow Cells ◽  
Response To Therapy ◽  
Poor Responders ◽  
Met Receptor ◽  
Potential Target ◽  
Marrow Cells

Abstract Despite progress in the treatment of Multiple Myeloma (MM), it is still an incurable disease with average survival of 3–4 years. Because MM is often resistant to conventional therapies, new treatment strategies are necessary. The presence of elevated HGF (Hepatocytic Grow Factor) expression has been well documented in multiple myeloma. The c-met oncogene has been shown to be present in MM cell lines at the mRNA and protein level. Some data suggested that this axis could be responsible for proliferation and inhibition of apoptosis in MM cells. In this study we have analyzed c-met expression in 15 patients with (MM) before and after treatment. Seven of these pts responded well and eight pts responded poorly to the employed therapy. All 15 pts were c-met positive before therapy. Bone marrow cellularity of patients who responded well was 76% before (range: 10% – 100%) and 46% after treatment (range: 40% – 60%). In this group plasmocyte infiltration of bone marrow consisted of 59% before (range: 10% – 80%) and 9% after chemotherapy (range: 0% – 20%). Five of them had undetectable c-met positive cells among bone marrow cells after treatment. In the group of poor responders cellularity of bone marrow was 40% (range: 20% – 70%) before treatment and 46% (range: 20% – 70%) after therapy. Plasmocytes consisted of 20% (range: 10% – 50%) of bone marrow cells before and 44% (range: 10% – 90%) after treatment. All patients in this group had cells positive for c-met receptor after therapeutic regiment. This results suggested that c-met-HGF axis might be a good target for alternative therapy in MM. We looked for potential therapeutics that interferes with this axis and we found that geldanamycin (GA) has been shown to decrease expression of c-met at the protein level in several different cell types. Using inhibitors that belongs to geldanamycin family (GA, 17AAG and 17DMAG) we treated MM cell lines and primary sample. We found that these molecules strongly inhibited expression of c-met in both MM cell lines and patients sample as assessed by western blot analysis. We also tested the influence of these inhibitors on proliferation of MM cells. We found that 100nM dose of GA and 17DMAG inhibited growth of MM cell lines by 80% and 100nM dose of 17AAG inhibited growth of these cells by 20%. Primary cells were more resistant to treatment but we still obtained 30% inhibition with GA and 17DMAG. 17AAG was ineffective and proliferation decreased by less than 10%. Grow inhibition was probably not only due to c-met-HGF axis blockade because these molecules also inhibit other proteins (AKT, RAF). In our experiments we have shown that the level of c-met expression correlates with response to therapy. Patients who respond well had substantially decreased number of c-met positive plasmocytes after chemotherapy in comparison to poor responders. We have also showed that drugs that block c-met-HGF axis could be used in treatment of MM. These drugs could potentially inhibit cells proliferation, increase apoptosis and disrupt MM cells interaction with bone marrow environment. Based on these data we postulate that the c-met receptor is a potential target for MM therapy especially in patients who do not respond to the first line of treatment.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

Fully Retargeted Oncolytic Measles Virus for Multiple Myeloma Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5474-5474
Author(s):  
Horst D. Hummel ◽  
Gaby Kuntz ◽  
Takafumi Nakamura ◽  
Axel Greiner ◽  
Stephen J. Russell ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Cell Lines ◽  
Plasma Cell ◽  
Measles Virus ◽  
Plasma Cells ◽  
Treatment Modalities ◽  
Single Chain ◽  
Gfp Expression ◽  
Frequent Relapses

Abstract Multiple Myeloma (MM) is a disseminated plasma cell malignancy with approximately 14,600 new cases diagnosed in the USA annually. Despite recent progress in current therapeutical options the median survival is 3 to 5 years and cure is extremely rare. Therefore the evaluation of new treatment modalities for MM is highly warranted. An attractive approach to treat Myeloma with a minimum of undesired side effects is the use of a tumour antigen specific for MM cells. Wue-1, a monoclonal antibody binds very selectively normal and malignant plasma cells (50 of 51 MM samples, 14 of 15 immunocytoma and 13 of 13 MALT type lymphomas with plasma cell differentiation were Wue-1 positive, normal tissue including hematopoietic cells were negative) and offers the possibility to define MM cells as targets. The tool for selective killing of MM cells recognized by Wue-1 monoclonal antibody is in this study the measles virus vaccine strain Edmonston B in an ablated variant (MV-Wue) which no longer binds the usual measles receptors CD46 and CD150 (SLAM) expressed on almost every human cell type displaying a single-chain antibody (scFv) derived from the monoclonal Wue-1-antibody which has been tethered to the C-terminus of the H protein to restrict and retarget its interaction to malignant plasma cells especially MM cells. In addition, MV-Wue encodes EGFP facilitating the read out of infected cells. To determine if the fully retargeted MV-Wue would be able to infect MM cell lines and primary MM cells selectively an array of infection assays were performed using the MM cell lines U266 as well as primary CD138 positive MM cells expressing the Wue-1 antigen as expected targets and CD138 negative cells and normal B cells as controls negative for Wue-1. In these experiments selective infections of the MM cell line and primary MM cells were observed whereas the control cells were not infected with MV-Wue. In all cell types GFP expression indicating replicative infection correlated with the expression of the Wue-1 antigen determined by FACS. Infection experiments performed in the presence of monoclonal Wue-1 antibody showed a decreased GFP expression of about 78% in CD138 positive MM cells demonstrating specificity of the infection by MV-Wue. These results indicate that the engineered virus can be a safe and potential curative oncolytic agent to face the main problem in Multiple Myeloma which is responsible for frequent relapses, the minimal residual disease (MRD).

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2056-2056 ◽  
Author(s):  
Chantiya Chanswangphuwana ◽  
Michael P. Rettig ◽  
Walter Akers ◽  
Deep Hathi ◽  
Matthew Holt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Molecular Diagnostic ◽  
Fluorescent Intensity ◽  
Variable Expression ◽  
Rpmi 8226

Abstract Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM. Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study. Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection. Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals PIM Kinase Inhibition Decreases the Proangiogenic Properties of Multiple Myeloma Cells and Affects the Metabolic State of the Vascular Endothelium

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Filip Garbicz ◽  
Anna Szumera-Ciećkiewicz ◽  
Joanna Barankiewicz ◽  
Dorota Komar ◽  
Michał Pawlak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Cell Death ◽  
Tumor Cell ◽  
Cell Lines ◽  
Fold Increase ◽  
Pim Kinases ◽  
Rpmi 8226

The development and progression of multiple myeloma (MM) depend on the formation and perpetual evolution of an immunosuppressive and hypervascular bone marrow microenvironment. MM undergoes an angiogenic switch during its early progression stages and initiates the secretion of proangiogenic proteins, such as VEGFA and Galectin-1. Following their engagement with the VEGF receptor 2 on the surface of the endothelium, quiescent endothelial cells (ECs) rapidly switch to an activated state, thus gaining the ability to create sprouts, migrate and proliferate. However, chronic angiogenic stimulation results in the formation of a dense and leaky network of pathological vessels, which in the case of MM also serves as a major source of prosurvival paracrine signals. Since PIM kinases are known modulators of cytokine signaling, owing to their ability to activate NFκB, JAK/STAT and mTOR pathways, we analyzed the expression pattern of PIM1, PIM2 and PIM3 in multiple myeloma bone marrow samples using immunohistochemistry. We found that both MM cells as well as myeloma-associated ECs exhibit a significantly higher PIM3 expression than their normal bone marrow counterparts. Since the role of PIM kinases in the vascular compartment of the tumor microenvironment is currently unknown, we decided to explore the proangiogenic functions of PIM kinases using in vitro MM and EC model cell lines. 3 MM cell lines (RPMI 8226, MM1.s, U266), immortalized bone marrow ECs (HBMEC-60) and human umbilical vein ECs (HUVECs) were used for the experiments. Primary MM cells were obtained from MACS-separated bone marrow aspirates. Chemical blockade of PIM kinase activity was achieved using the pan-PIM inhibitor SEL24/MEN1703. The compound decreased the viability of MM cell lines with IC50 in the submicromolar range, induced G2 cell cycle arrest and apoptosis. Moreover, SEL24/MEN1703 induced apoptosis in primary MM cells, even when cocultured with the CD138- bone marrow fraction. PIM inhibitor treatment inhibited the phosphorylation of mTOR substrates S6 and 4EBP1, STAT3/5, as well as RelA/p65. Consequently, we observed markedly decreased VEGFA and Gal-1 levels in SEL24/MEN1703-treated MM cells. When cultured together, separated by a permeable transwell membrane, both RPMI 8226 cells, as well as ECs, exhibited a 2-fold increase in proliferation rate. This effect was completely blocked by a 2-day treatment with a PIM inhibitor. Exposure of ECs to recombinant VEGFA (10ng/ul) or MM supernatant resulted in an increase in VEGFR2 Y1175 phosphorylation level and induction of PIM3 expression. Increased MYC activity is a hallmark of VEGF-dependent endothelial activation and is necessary to support the creation of new vessels. Since the PIM3 promoter region contains putative MYC-binding sites (E-boxes), we checked if PIM3 induction depends on MYC in ECs. MYC silencing using siRNA resulted in an 88% lower PIM3 expression than the non-targeting siRNA. One of MYC's main tasks during angiogenesis is the stimulation of cellular ATP synthesis to meet the energy demands created by the dynamic remodeling of the actin cytoskeleton. Surprisingly, PIM inhibition decreased the total ATP content in ECs by 25%, thus disrupting the energetic homeostasis, as evidenced by a 9.6-fold increase in phosphorylated AMPK T172 levels. Furthermore, SEL24/MEN1703-treated ECs were depleted of higher-order actin structures necessary for efficient angiogenesis, such as actin stress fibers, membrane ruffles and lamellipodia. In consequence, PIM kinase inhibition decreased proliferation, migration and formation of new vessel-like structures in Matrigel by ECs. Collectively, our data demonstrate that PIM inhibition induces MM cell death and abolishes important tumor cell-ECs interactions. In addition, we show that PIM3 is overexpressed in MM tumor endothelial cells and PIM inhibition disrupts the activation state in in vitro cultured ECs. Hence, targeting PIM kinases may represent an efficient approach to induce tumor cell death and to block angiogenesis in MM. RNA-sequencing studies on the downstream effectors of PIM3 are currently ongoing in order to unravel the molecular mechanism behind the observed effects. Figure Disclosures Brzózka: Ryvu Therapeutics: Current Employment. Rzymski:Ryvu Therapeutics: Current Employment. Tomirotti:Menarini Ricerche: Current Employment. Lech-Marańda:Roche, Novartis, Takeda, Janssen-Cilag, Amgen, Gilead, AbbVie, Sanofi: Consultancy; Roche, Amgen, Gilead: Speakers Bureau. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

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