MEDI2228, a Novel Bcma Antibody-PBD Conjugate, Sensitizes Human Multiple Myeloma Cells to NK Cell-Mediated Cytotoxicity and Upregulates CD38 Expression in MM Cells

MEDI2228, an antibody-drug conjugate (ADC) comprised of an anti-BCMA antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine dimer, is currently under clinical development for the treatment of human multiple myeloma (MM) (NCT03489525). MEDI2228 induces DNA damage responses (DDR) prior to apoptosis, as demonstrated by phosphorylation of ATM/ATR, CHK1/2, and gH2AX in MM cells regardless of p53 status and responsiveness to current MM therapies including bortezomib and IMiDs. Since activation of DDR alters expression of ligands for NKG2D receptors critical for NK-mediated immune surveillance, we here examined whether the ATM/ATR-CHK1/2 signaling cascades activated by MEDI2228 treatment would increase NKG2D ligands in MM cells. Using real-time quantitative RT-PCR and flow cytometry analysis, we found that treatment with MEDI2228 increased the expression of major histocompatibility complex (MHC) class I chain-related proteins A and B (MICA/B) in MM cell lines (n>5) and CD138+ MM cells from patients with relapsed and refractory disease (n=4). In addition, expression of the MHC class I molecules/NKG2D ligands ULBP-1, -3, -2/5/6 increased following MEDI2228 treatment. Next, we evaluated NK cell-mediated lysis of MM target cells (n>3) with or without pretreatment with MEDI2228 and found increased NK cell-mediated lysis of MEDI2228-pretreated vs control MM cells in an effector-target ratio-dependent manner. In parallel, we examined whether MEDI2228 stimulates STAT1- and IFN-related signaling pathways since they are activated by DDR and play a crucial role in innate and adaptive immunity. We found that MEDI2228 treatment significantly increases phosphorylation of STAT1 in H929 and its derived IMiD-resistant cells, and further augments expression of IFN-induced genes (IFITs), IFIT1, 2, 3, and 5, which have been shown to inhibit proliferation and promote apoptosis in cancer cells. Significantly, CD38 is upregulated by MEDI2228 treatment, with increased mRNA expression as well as membrane expression detected by flow cytometry in MM cell lines and MM cells from newly diagnosed and refractory patients (n=5). Consequently, MEDI2228-pretreated MM cells (n>3) are more susceptible to NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) by daratumumab, which targets CD38. Taken together, our data show that MEDI2228-induced DDR primes MM cells to NK cell-mediated cytotoxicity by increasing expression of MICA/B in MM cells to enhance binding and activating NK cytolytic activity. Simultaneously, MEDI2228 induces IFN-stimulated genes, including CD38, resulting in enhanced MM cell lysis by daratumumab. These results indicate additional mechanisms of anti-MM activity for MEDI2228 and suggest that a combination of MEDI2228 and anti-CD38 mAbs may further improve outcome for MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Janssen: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy. Anderson:Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau.

Download Full-text

A Novel Mechanism of Action of Bortezomib: Proteasome Inhibition Down-Regulates HLA-Class I on Myeloma and Enhances NK Cell-Mediated Lysis.

Blood ◽

10.1182/blood.v110.11.1525.1525 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1525-1525

Author(s):

Jumei Shi ◽

Guido J. Tricot ◽

Priyangi A. Malaviarachchi ◽

Tarun K. Garg ◽

Susann M. Szmania ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Cell Activity ◽

Hla Class I ◽

Class I ◽

Target Cells ◽

Down Regulation

Abstract Introduction: Natural killer (NK) cell activity is regulated by a dynamic balance between inhibitory and activating receptors that recognize ligands on target cells. Human leukocyte antigen (HLA)-class I, particularly HLA-C and -Bw4 molecules, are key ligands transmitting inhibitory signals to NK cells. NK cells avidly lyse tumor cells that do not display such inhibitory KIR-ligands. The proteasome is responsible for the generation of peptides that bind to and stabilize class I molecules at the cell surface. We hypothesized that bortezomib, a partial proteasome inhibitor that is clinically approved for the treatment of refractory/relapsed myeloma (MM), could reduce HLA expression on MM cells and thus enhance NK cell-mediated cytotoxicity. Methods: HLA-class I or HLA-C expression was assessed using flow cytometry, after gating on AnnexinV/PI double negative cells, and/or confocal microscopy. Expression of other proteins was measured by flow cytometry using specific mAb. NK cell-mediated lysis of myeloma cells was measured by 51Cr-release. Results: Bortezomib at clinically attainable concentrations down-regulated HLA-class I expression on MM cells in a time- and dose-dependent fashion. Reduction of class I post-10 nM bortezomib treatment was observed in all myeloma cell lines tested (n=10), by a median of 49% (range: 19–66%). A similar decrease of HLA-class I was obtained in 10–50 nM bortezomib treated primary MM cells (n=6). Bortezomib significantly enhanced the sensitivity of MM cells to allogeneic and autologous NK cell-mediated lysis. Further, the level of reduction in HLA-class I expression correlated well with increased susceptibility to lysis by NK cells. The level of down-regulation of HLA-class I induced by bortezomib was reproduced by incubating MM cells with HLA-blocking antibody and resulted in equipotent enhancement of NK cell-mediated lysis (Figure 1). The extent of HLA-class I down-regulation by bortezomib was therefore biologically relevant. Down-regulation of HLA-class I was also observed in vivo on purified MM cells 48 hours after a single dose of bortezomib, by a median of 47% (range: 16–63%, n=6, P= .002). HLA-C expression (the principal NK cell inhibitory ligand) was rescued by exogenous provision of HLA-C binding peptides providing a mechanistic explanation for the effect of bortezomib on HLA-class I expression. Finally, we did not observe bortezomib-mediated enhancement of NK cell-mediated lysis of myeloma through receptors other than the KIR receptor family, including tumor necrosis factor related apoptosis-inducing ligand, NKG2D and natural cytotoxicity receptors. HLA-class I down-regulation was not observed in renal cell and breast carcinoma cell lines, which is in keeping with the remarkable activity of bortezomib in myeloma. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells. Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis

Download Full-text

A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

Journal of Experimental Medicine ◽

10.1084/jem.185.12.2053 ◽

1997 ◽

Vol 185 (12) ◽

pp. 2053-2060 ◽

Cited By ~ 153

Author(s):

Ennio Carbone ◽

Giuseppina Ruggiero ◽

Giuseppe Terrazzano ◽

Carmen Palomba ◽

Ciro Manzo ◽

...

Keyword(s):

Nk Cells ◽

Mhc Class I ◽

Nk Cell ◽

Cd40 Ligand ◽

Class I ◽

Target Cells ◽

Ligand Interaction ◽

Effector Functions ◽

Nk Cell Cytotoxicity ◽

Dependent Pathway

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

Download Full-text

Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

Journal of Experimental Medicine ◽

10.1084/jem.178.4.1321 ◽

1993 ◽

Vol 178 (4) ◽

pp. 1321-1336 ◽

Cited By ~ 117

Author(s):

V Litwin ◽

J Gumperz ◽

P Parham ◽

J H Phillips ◽

L L Lanier

Keyword(s):

Nk Cells ◽

Mhc Class I ◽

Target Cell ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Target Cells ◽

Clonal Heterogeneity ◽

Multiple Alleles ◽

Wide Range

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

A Fully Human Antagonist Anti-CD40 Antibody Triggers Significant Antitumor Activity Against Human Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.2414.2414 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2414-2414

Author(s):

Yu-Tzu Tai ◽

Xian-Feng Li ◽

Xia Tong2 ◽

Laurence Catley ◽

Daniel Santos ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Dependent Manner ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Growth And Survival ◽

Human Multiple Myeloma ◽

Dose Dependent

Abstract We previously demonstrated that CHIR-12.12, a fully human anti-CD40 mAb (IgG1) generated in XenoMouseÒ mice (Abgenix, Inc), blocks CD40/CD40 ligand (CD40L) interactions and has more potent anti-lymphoma activity than Rituximab both in vivo and in vitro (abstract #2386, ASH, San Diego, Dec. 2003). In this study, we assess the efficacy of CHIR-12.12 against human multiple myeloma (MM) using CD40-expressing MM cell lines and purified CD138+ patient cells. CHIR-12.12 binds to purified CD138+ MM cells in >80% (10/12) of patient samples, as measured by flow cytometry: the mean fluorescence intensity (MFI) range was 1 to 20 for CHIR-12.12 vs 0.2–0.9 for control human IgG1. We next examined the antagonist activity of CHIR-12.12 in MM cells. CHIR-12.12 blocked CD40L-mediated proliferation of CD40-expressing MM lines and purified CD138+ patient cells from 2 MM patients in a dose-response manner. In contrast, CHIR-12.12 alone did not alter constitutive MM cell proliferation. Immunoblotting analysis demonstrated that PI3-K/AKT, NF-kB, and ERK activation induced by hCD40L in the 12BM MM cell line was significantly inhibited by CHIR-12.12 (5 μg/ml). Adhesion of MM cells to bone marrow stromal cells (BMSCs) confers growth and survival benefit for tumor cells. Since CD40 activation, either by stimulatory mouse anti-CD40 mAb G28.5 or formaldehyde-fixed CHO cells expressing hCD40L, induces MM cell adhesion to fibronectin (FN) or BMSCs, we next asked whether antagonist CHI12.12 abrogates this process. CHIR-12.12 inhibited CD40L-induced adhesion of MM cell lines to FN in a dose dependent manner (0.001-10 μg/ml), whereas control human IgG did not. Moreover, CHIR-12.12 (1 μg/ml) blocked hCD40L-induced adhesion of freshly isolated patient MM cells to BMSCs. Adhesion of MM cells to BMSCs induces IL-6 secretion, an important growth and survival cytokine for MM cells, and treatment of MM cells with hCD40L further augmented adhesion-induced IL-6 secretion. Conversely, pretreatment of CD40-expressing MM cell lines with CHIR-12.12 significantly decreased IL-6 secretion triggered by coculture of MM cells with BMSCs. We next examined whether CHIR-12.12 stimulates antibody-dependent cellular cytotoxicity (ADCC) against CD40-expressing MM cells. Human peripheral blood mononuclear cells and purified NK cells (CD56+CD3−) were used as effector cells. CHIR-12.12 triggered MM cell lysis in a dose dependent manner, as measured in CD40-expressing MM cell lines. The maximum specific lysis of 20–70 % was achieved at 10 μg/ml concentration of CHIR-12.12. CHIR-12.12 mediated lysis was specific to CD40-expressing MM cells, as CHIR-12.12 did not induce ADCC against CD40-negative MM cells. Importantly, CHIR-12.12 induced ADCC against CD138+ cells isolated from 2 MM patients. These results provide preclinical rationale for clinical evaluation of CHIR-12.12 with the goal of improving patient outcome in MM.

Download Full-text

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 ◽

10.1182/blood.v108.11.3460.3460 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3460-3460 ◽

Cited By ~ 1

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.

Download Full-text

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3457.3457 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3457-3457 ◽

Cited By ~ 3

Author(s):

Eric D. Hsi ◽

Roxanne Steinle ◽

Balaji Balasa ◽

Aparna Draksharapu ◽

Benny Shum ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Target Cells ◽

Dependent Manner ◽

Myeloma Cells ◽

Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

Download Full-text

Antitumor Effects and Mechanisms of Curcumin On Human Multiple Myeloma Cell Lines.

Blood ◽

10.1182/blood.v114.22.4896.4896 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4896-4896

Author(s):

Qingxian Bai ◽

Qifa Liu

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Synergistic Effect ◽

Cell Lines ◽

Myeloma Cell ◽

Dependent Manner ◽

Antitumor Effects ◽

Low Concentration ◽

Multiple Myeloma Cell ◽

Human Multiple Myeloma

Abstract Abstract 4896 BackgroundF Multiple myeloma(MM) is a malignant plasma disease, which is characterized as high relapse rate and high resistance to chemotherapy. Curcumin is a polyphenol derived from the rhizome of Curcuma spp. It possesses diverse pharmacologic actions, such as antitumor, anti-inflammatory,anti- oxidation properties .Curcumin has the property of inhibit multiple tumor cell lines, in which included multiple myeloma cell. The real mechanism is not completely clear yet. We explored the mechanisms of curcumin on human multiple myeloma cell lines (RPMI8226 and H929), and investigated whether the combination of curcumin and adriamycin(Adr) has a synergistic effect. MethodsF The effect of curcumin on proliferation of RPMI8226 and H929 was observed with MTT assay. The synergetic effect of curcumin and Adr was analyzed by median-effect principle. Cell cycle distribution and apoptosis were studied with flow cytometry. Expression of surviving, bcl-2, bax mRNA was detected by RT-PCR. ResultsF Curcumin could inhibit the proliferation of RPMI8226 and H929 cells in a time- and dose-dependent manner. The IC50 values for RPMI8226 and H929 cell line were 12.15 μmol/L,17.24μmol/L respectively. The combination of curcumin and Adr showed synergistic effect even at low concentration of Adr. Apoptotic ratio of treated cells was significantly higher than untreated controls (36.9% vs 10.6%, p<0.05). Cells treated with curcumin showed cell cycle arrest at G2/M phase. Curcumin upregulated expression of survivin, bcl-2, while bax mRNA was significantly downregulated. ConclusionF Curcumin could suppress the proliferation of multiple myeloma cells and induce apoptosis. Adr combining with curcumin can show synergistic effect at low concentration of Adr. The mechanism of curcumin's antitumous effect might be related to down-regulation of surviving, bcl-2 mRNA and up-regulation of bax mRNA. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Monocarboxylate Transporters Play an Important Role in Aerobicglycolysis in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.1821.1821 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1821-1821

Author(s):

Shiho Fujiwara ◽

Naoko Wada ◽

Yawara Kawano ◽

Hiromichi Yuki ◽

Yutaka Okuno ◽

...

Keyword(s):

Energy Source ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Cancer Cells ◽

Cell Lines ◽

Aerobic Glycolysis ◽

Lactate Production ◽

Monocarboxylate Transporters ◽

Dependent Manner ◽

Growth And Survival

Abstract Abstract 1821 Introduction It has been reported that cancer cells utilize glycolysis pathway (non-oxidative breakdown of glucose) even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis) that ultimately leads to produce lactate. We reported in the last ASH meeting that aerobic glycolysis is up-regulated in multiple myeloma (MM) cells in patients with high serum LDH levels and aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Here we report an importance of lactate transporter for the growth and survival of MM cells. Lactate, produced from pyruvate by lactate dehydrogenase A (LDHA), is known as an important energy source for solid tumor cells and is associated with tumor angiogenesis and chemo-resistance (Pinheiro, C., et al. J Bioenerg Biomembr. 44:127–139, 2012). On the other hand, LDHB converts lactate to pyruvate, thus negatively regulating lactate production. It is known that lactate is pumped out through monocarboxylate trasnporter, MCT4, while MCT1 mainly imports lactate to inside of cells. However, roles of MCT1 and MCT4 in MM cells remain to be elucidated. We here investigated the roles of these two molecules in the growth and survival of MM cells. CD147, a purported chaperone protein for MCT1, was also examined. Methods Six MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were employed. Six genes associated with glycolysis, i.e., LDHA, LDHB, MCT1-4, were examined using real time PCR analysis. Expressions of MCT1 and MCT4 were analyzed with western blotting. Expression of CD147 was investigated by flow cytometry. Lactate production into culture supernatants of MM cell lines were analyzed by using a lactate analyzer. An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells. AnnexinV/PI stained cells was analyzed by flow cytometry to quantify cytotoxicity. MCT1-expression was inhibited by using siRNA. Dichroloacetate (DCA), an inhibitor of PDK1, was utilized for inhibiting glycolysis. Results Accumulation of lactate was found in the supernatants of MM cell lines as cell density increased. Transporters of lactate, MCT1, MCT4 and CD147, were found in most MM cell lines at various levels, suggesting that transportation of lactate occurs through membrane of MM cells. To examine the role of lactate as a growth promotion factor, lactate was exogenously supplemented to KMS-12-PE cells. Interestingly, expressions of MCT1 and LDHB genes increased by the addition of lactate while those of MCT4 and LDHA only moderately changed (Fig. 1), suggesting that lactate was imported to cells through MCT1, then converted to pyrvate by LDHB. These results raised a possibility that lactate is utilized by MM cells as a growth factor. To examine the possibility, CHC, an inhibitor of MCT1, was supplemented to MM cell cultures. Interestingly, CHC induced apoptosis in MM cells in a dose dependent manner (Fig. 2). Moreover, inhibition of MCT1 gene by siRNA showed significant induction of apoptosis (Fig. 3), strongly suggesting that MCT1 plays a crucial role for survival of MM cells. Finally, we found a significant increase in the apoptosis of MM cells when CHC and DCA were simultaneously added in the culture (Fig.4), suggesting that MCT1 functions independently from glycolysis per se and that CHC and DCA act additively in starving lactate within MM cells. Conclusion Our results suggest that lactate is actively transported through monocarboxylate transporters. Given the results that exogenous lactate production increased MCT1 and LDHB expression, lactate should play a role as a regulator of lactate transportation and glycolysis as well as an important energy source. Because we found significant amount of lactate was produced from stromal cells obtained from MM patients, lactate may be supplied not only from MM cells themselves but also from micro-environment. Our finding that inhibition of MCT1 leads to cell death suggests that MCT1 could be a potential novel target molecule in MM therapy that could be stratified in combination with glycolysis inhibitor. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Phospholipase C-γ2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells

Blood ◽

10.1182/blood-2005-06-2428 ◽

2006 ◽

Vol 107 (3) ◽

pp. 994-1002 ◽

Cited By ~ 86

Author(s):

Anouk Caraux ◽

Nayoung Kim ◽

Sarah E. Bell ◽

Simona Zompi ◽

Thomas Ranson ◽

...

Keyword(s):

Innate Immunity ◽

Nk Cells ◽

Phospholipase C ◽

Mhc Class I ◽

Nk Cell ◽

Class I ◽

Target Cells ◽

Cell Cytotoxicity ◽

Infected Cells ◽

Nk Cell Cytotoxicity

AbstractPhospholipase C-γ2 (PLC-γ2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-γ2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-γ2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-γ2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-γ on stimulation with IL-12 was normal. Plcg2-/- mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection. Our results suggest that exocytosis of cytotoxic granules, but not cellular polarization toward targets, depends on intracellular calcium rise during NK cell cytotoxicity. In vivo, PLC-γ2 regulates selective facets of innate immunity because it is essential for NK cell responses to malignant and virally infected cells but not to bacterial infections.

Download Full-text

Impacts of a Proliferation-Inducing Ligand on Current Therapeutic Monoclonal Antibody-Induced Cytotoxicity Against Human Multiple Myeloma Cells

Blood ◽

10.1182/blood-2019-130927 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3105-3105 ◽

Cited By ~ 1

Author(s):

Liang Lin ◽

Shih-Feng Cho ◽

Kenneth Wen ◽

Tengteng Yu ◽

Phillip A Hsieh ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Membrane ◽

Nk Cell ◽

Cell Lysis ◽

Advisory Board ◽

Dependent Manner ◽

Effector Cells ◽

Human Multiple Myeloma ◽

Dose Dependent

A proliferation inducing ligand (APRIL) is a natural ligand for B cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), two receptors overexpressed in human multiple myeloma (MM) patient cells. Specifically, BCMA is highly expressed in plasma cells of all MM patients and BCMA-based immunotherapies has recently shown impressive response rates in patients with relapsed and refractory diseases. APRIL, mainly secreted by myeloma-supporting bone marrow (BM) accessory cells, i.e., macrophages, osteoclasts (OC), promotes MM cell progression in vitro and in vivo. It further induces survival and function of regulatory T cells (Treg) via TACI, but not BCMA, to support an immunosuppressive MM BM microenvironment (Leukemia. 2019;33:426). Here, we study effects of APRIL in current immunotherapies in MM and determine whether APRIL influences antibody-dependent cellular cytotoxicity (ADCC) induced by therapeutic anti-BCMA (J6M0) or anti-CD38 (daratumumab) mAbs via FcR-expressing immune effector cell-dependent mechanisms. Using anti-human IgG1 to detect J6M0 binding to the cell membrane BCMA, we first showed that APRIL, in a dose-dependent manner (31-500 ng/ml), competed with J6M0 for binding to BCMA. Such effects were inhibited by the blocking anti-APRIL monoclonal antibody (mAb) (Apry-1-1), as confirmed by flow cytometry and confocal microscopy. APRIL still inhibited J6M0 binding to BCMA at 4°C, arguing against that APRIL induces shedding of BCMA receptor. Using PE labeled anti-FLAG to detect APRIL-FLAG bindings to MM cell surface BCMA, J6M0 (0.25-4 µg/ml) did not alter APRIL binding to BCMA following 2h or 1d incubation. High concentrations of J6M0 (>10 µg/ml) only blocked ~50% of APRIL (0.2 µg/ml)-induced NFκB activity as determined by specific DNA binding assays, indicating that APRIL-induced signaling cascade via BCMA or TACI in MM cells is not completely blocked by J6M0. In parallel, data analysis using mRNA-seq identified 594 or 355 differentially expressed genes (Log2-Fold-change > 1.5 and adjusted p < 0.05) in APRIL- and BCMA-overexpressed RPMI8226 MM cell transfectants, respectively, when compared with control parental cells. KEGG and Reactome pathway enrichment analysis further defined that these differentially expressed genes are enriched in cell adhesion, migration, chemokine signaling pathways, and JAK/STAT signaling pathways, in addition to proliferation and survival in MM cells. We next asked whether overnight treatment with APRIL in MM cell lines decreased their baseline lysis by FcR-expressing effector cells, i.e., NK, monocytes. In a dose-dependent manner, APRIL (10-200 ng/ml) downregulated baseline MM cell lysis mediated by these effector cells. Importantly, in a similar fashion, ADCC was decreased against all APRIL-treated vs control MM cell lines induced by J6M0 or daratumumab. Conversely, blocking anti-APRIL mAbs reverted APRIL-suppressed cytotoxicity against MM cells induced by J6M0 or daratuzumab. These results were validated by decreased J6M0-induced NK cell degranulation following co-incubation with APRIL-treated vs control MM cells. In contrast, anti-APRIL neutralizing mAbs specifically blocked APRIL-inhibited NK cell membrane CD107a expression. Furthermore, co-cultures with MM-supporting OCs or macrophages decreased ADCC against MM cells by NK cells; conversely the neutralizing anti-APRIL mAb significantly blocked APRIL-reduced MM cell lysis by J6M0- or Daratumumab. Finally, APRIL reduced J6M0-induced patient MM cell lysis when freshly isolated BM mononuclear cells from MM patients (n=10) were incubated with NK cells from the same individual. Anti-APRIL mAbs still blocked APRIL blockade in J6M0-induced autologous patient MM cell lysis. Taken together, our data further indicate that therapies directed at the APRIL/BCMA and APRIL/TACI axes may simultaneously target MM cells and counteract APRIL-reduced MM cell lysis induced by therapeutic mAbs targeting MM cells. These results thus support combination strategies of blocking APRIL mAbs with BCMA- or CD38-directed immunotherapies to further overcome MM cell-induced immunosuppressive BM microenvironment, thereby enhance Disclosures Munshi: Abbvie: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder .

Download Full-text