International Journal of Myeloma 10(1): 1–7, 2020
©Japanese Society of Myeloma

ORIGINAL

Notch1–JAG1 signaling induces aggressive myeloma cell behaviors
Mariko ISHIBASHI1, Keima UEDA2, Yoichi IMAI3, Koiti INOKUCHI2,
Rimpei MORITA1 and Hideto TAMURA2The Notch signaling pathway plays a crucial role in the tumor microenvironment. We examined the expression of
Notch receptors (Notch1, Notch2, Notch3, and Notch4) and its ligands (JAG1, JAG2, DLL1, DLL3, and DLL4) in
multiple myeloma (MM) cells and the functions of Notch signaling in disease progression. NOTCH1, NOTCH2, and
JAG1 mRNA were highly expressed in 17 MM cell lines and primary MM cells from 28 patients, although mRNA levelsof NOTCH1 were significantly lower than those of NOTCH2. However, anti-Notch1 antibody inhibited MM cell proliferation and increased the percentage of bortezomib-induced apoptotic MM cells as compared with isotype control
and cells cultured with anti-Notch2 antibody. Furthermore, Notch1+ MM cells showed greater proliferative potential
and decreased drug susceptibility compared with Notch– cells. Aggressive disease behaviors in Notch1+ MM cells
were suppressed by anti-JAG1 antibody. Furthermore, gene expression in Notch1+ cells showed upregulation of p21
and CCND1/2 and downregulation of apoptotic genes compared with those in Notch– cells. Those results demonstrate that the Notch1–JAG1 signaling pathway confers a high malignant potential, suggesting that its Notch
signaling may be specifically associated with MM disease progression.
Key words:

multiple myeloma, Notch signaling, Notch1, JAG1Introduction

over, the programmed death ligand 1 (PD-L1) on MM cells is
involved in aggressive myeloma behaviors including cell pro-Multiple myeloma (MM) is an incurable hematologicalliferation capacity and drug resistance, and the interaction ofmalignancy characterized by bone marrow (BM) infiltration of

PD-L1 with programmed cell death 1 (PD-1) molecules induces

abnormal plasma cells, and most MM patients progress fromdrug resistance in MM cells by antiapoptotic responses throughan indolent (slowly developing) to a virulent (aggressive) form

the Akt signaling pathway [5, 6]. Therefore, the cell prolifera-over many years [1, 2]. During MM progression, abnormaltion, drug resistance, and survival of MM cells are strongly

plasma cells (MM cells) gradually acquire malignant potential

facilitated by the interactions among several cytokines pro-through multistep genetic and BM microenvironmental

duced by MM and stromal cells as well as with other cell com-changes [2, 3]. Our previous study demonstrated that theponents in the BM niche [3, 7]. Increasing understanding of­signaling lymphocytic activation molecule family 3 (SLAMF3;

the mechanism of MM malignant formation in the BM niche

also known as CD229 or Ly9) on MM cells transmits MAPK/ERK

is providing clues for the development of novel therapeutic

signals mediated by self-ligand interaction between MM cells

strategies.

and induces a high malignant potential in MM [4]. More­

The Notch signaling pathway, which is one of the most­commonly activated signaling pathways in various cancers,

Received January 11, 2020, accepted February 20, 2020
1
Department of Microbiology and Immunology, Nippon Medical
School, Japan2
Department of Hematology and Immunology, Nippon Medical
School, Japan3
Department of Hematology and Oncology, IMSUT Hospital, The
Institute of Medical Science, The University of Tokyo, Japan
Corresponding author: Hideto TAMURA, MD, PhD
Department of Hematology, Nippon Medical School, 1-1-5 Sendagi,
Bunkyo-ku, Tokyo 113-8603, Japan
E-mail: tam@nms.ac.jp

plays an important role in tumorigenesis as a major actor inboth cancer cells and in each of the components of the tumor
microenvironment [8, 9]. The Notch family has four trans­
membrane receptors, i.e., Notch1–4, that bind its ligands, i.e.,
Jagged1 (JAG1), JAG2, and Delta-like 1 (DLL1), DLL3, and DLL4,
resulting in the modulation of cell-fate determination, tissue
patterning, and morphogenesis. In the MM microenvironment,
Notch receptors are expressed on MM cells, BM stroma cells,
and osteoclasts, indicating that the activation of Notch signaling is involved in MM pathogenesis [10, 11]. In this study,
we focused on Notch signaling in MM disease progression.

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ISHIBASHI et al.

Thus, we examined the expression of Notch family molecules

plasma cells using TRIzol reagent (Thermo Fisher Scientific)

and their ligands in MM and the functions of Notch signaling

according to the manufacturer’s instructions. cDNA was syn-in MM progression.

thesized from total RNA, and then quantification of mRNA
using real-time PCR was performed [5, 6]. The relative expres-Materials and Methods

sion was calculated by the formula 2^(–delta Ct) × 1000. The
delta Ct value was calculated by subtracting Ct values (target

Human MM cell lines

gene – internal control gene); β-actin was used as an internal

Human MM cell lines were cultured in RPMI1640 medium(Wako Chemical Industries, Osaka, Japan) containing 10% fetal

control. The primer sequences are shown in Supplementary
Table S1.

bovine serum, 100 U/ml of penicillin, and 100 mg/ml of­streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at

Flow cytometry analysis

37°C under 5% CO2. KMS-5, KMS-11, KMS-18, KMS-20, KMS-27,

For flow cytometry (FCM) analysis, cells were suspended in

KMS-28PE, KMS-28BM, and KMS-34 were kindly provided by

50 μl of fluorescence-activated cell sorter (FACS) buffer (PBS

Dr. Takemi Otsuki (Kawasaki Medical School, Okayama, Japan).

with 5% bovine serum albumin and 10 mM sodium azide) andU266, RPMI8226, and MM.1S were obtained from the American

blocked with Human TruStain FcX (BioLegend). Then the cells

Type Culture Collection. OPM2 was purchased from DSMZ

were stained with PE-conjugated anti-Notch1 (clone #MHN1-(Braunschweig, Germany). The human MM cell lines MOSTI-1,

519) and anti-Notch2 antibody (clone #HMN2-35; BioLegend)-2, -4, -6, and -40 were established in our laboratory usingfor 30 min. Data acquisition was performed in a LSRFortess+–BM samples from MM patients [6]. Notch1 and Notch1 cell

X-20 flow cytometer with FACSDiva software version 8.0.1 andfractions were sorted using the FACSAria II cell sorter (BDanalyzed using Flowjo software (BD Biosciences).

­Biosciences, San Jose, CA, USA) after the cells were stainedwith PE-conjugated anti-Notch1.

Cell proliferation and apoptosis assay

Patient samples

FCM as described previously [5, 6]. To assess drug-induced

Bromodeoxyuridine (BrdU) analysis was performed using
BM samples were obtained from newly diagnosed MM

apoptosis, cells were exposed to melphalan and bortezomib

patients for diagnostic purposes at Nippon Medical Schoolfor 24 hours at concentrations optimal for inducing apoptosis,

after informed consent had been obtained according to theand then the cells were stained with annexin-FITC (BD Biosci-

Institutional Review Board-approved protocol. Mononuclear

ences) and propidium iodide (PI; Wako Chemical Industries) forcells (MNCs) were separated from BM samples with Histopaque

FCM analysis.

(Sigma-Aldrich, St. Louis, MO, USA) density centrifugation, andthen CD138+ plasma cells were purified from BM MNCs using
the Human CD138 Positive Selection Kit (Stemcell Technolo++

dim

Statistical analysis
The Student t test and Spearman’s rank moment correlation–

gies, Vancouver, Canada). CD38 CD138 and CD38 CD138

coefficient were used to analyze the data. P values of less thancells were sorted from BM MNCs of MM patients using the0.05 were considered to represent statistically significant dif-

FACSAria II cell sorter (BD Biosciences) after the cells were

ferences. Statistical analyses were performed using GraphPad

stained with FITC-conjugated anti-CD138 (clone #MI15) andPrism version 8.3.0 software (San Diego, CA, USA).

BV421-conjugated anti-CD38 (clone #HIT2; BioLegend, San

Results

Diego, CA, USA).

Expression of Notch family molecules and their ligands in MM

Reagents
Anti-human Notch1 (MAB5317; clone #527425), anti-Notch2

We first examined the expression levels of Notch family mol-

(MAB37351; clone #602845), and anti-JAG1 antibodies (MAB

ecules, which include four receptors, Notch1, Notch2, Notch3,

1277; clone #188331; R&D Systems, Minneapolis, MN, USA)and Notch4, and their receptors JAG1, JAG2, DLL1, DLL3, andwere used as blockade antibodies. Bortezomib and melphalan

DLL4 in 17 MM cell lines and primary MM cells from 28

were purchased from Selleck Chemicals (Houston, TX, USA),

patients. NOTCH1 and NOTCH2 mRNA expression was detectedreconstituted in dimethyl sulfoxide (DMSO), and stored atin MM cell lines and primary MM cells, but only very low–20°C until use.

expression of NOTCH3 and NOTCH4 was detected in MM celllines (Fig. 1A and 1B). Further, NOTCH2 mRNA expression wasmRNA expression analysis

higher than that of NOTCH1 mRNA (Fig. 1A and 1B). Although

Total RNA was extracted from MM cell lines and CD138+
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the expression level of NOTCH1 mRNA was low, it was detectedNotch1–JAG1 signaling pathway in myeloma

Figure 1. mRNA expression of Notch family molecules and ligand genes in MM cell lines and primary MM cells from patients. mRNA expression of
Notch family genes NOTCH1, NOTCH2, NOTCH3, and NOTCH4 and mRNA expression of ligand genes JAG1, JAG2, DLL1, DLL3, and DLL4 were
quantified using real-time PCR in MM cell lines (A and D) and primary MM cells from patients (B and E). (C) Cell-surface Notch1 and Notch2
expression in MM cell lines and primary MM cells analyzed using FCM. Solid line, staining with PE-conjugated antibody to Notch1 or Notch2;
filled area, staining with isotype-matched control Ig. The numerical values represent the percentage of Notch1/Notch2+ cells in each MM cell
culture.

on the cell surface in MM cell lines (Fig. 1C). On the other hand,

Aggressive myeloma behaviors associated with Notch1

JAG1 and JAG2 mRNA was highly expressed in MM cell lines,

molecules

but the expression levels of DLL1, DLL3, and DLL4 were low

We next investigated whether the cell proliferative potential(Fig. 1D). In primary MM cells, expression levels of JAG1 mRNAand drug resistance of MM cells were associated with Notch1

were significantly higher than those of JAG2 (Fig. 1E). The

or Notch2 molecule expression on the cells. BrdU incorpora-mRNA expression of NOTCH1 and NOTCH2 was not statisticallytion in both U266 and MM.1S cells was significantly decreased

correlated with its ligands JAG1 and JAG2 (data not shown).

after co-culture with anti-Notch1 antibody, but not after

The NOTCH1 mRNA level was significantly correlated only withco-culture with the isotype control and anti-Notch2 (Fig. 2A).

corrected calcium levels (r = 0.598, P = 0.0113), and highFurthermore, bortezomib-induced apoptosis was markedly

NOTCH1 mRNA levels in plasma cells from MM patients wereincreased by blockade of Notch1, but not of Notch2 (Fig. 2B).

not associated with poor survival times or other clinical

The mRNA of NOTCH1 and NOTCH2 was not changed by­characteristics (data not shown).

blocking of anti-Notch1 or anti-Notch2 antibodies in U266

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ISHIBASHI et al.

Figure 3. Cell proliferation and drug sensitivity of Notch1+ andNotch1– U266 cells. (A) Cell surface Notch1 expression in the
U266 cell line analyzed using FCM. BrdU incorporation (B), andmelphalan and bortezomib sensitivity (C) in Notch1+ andNotch1– U266 cells. The data are expressed as mean ± standard
deviation of triplicate experiments. *P < 0.05, **P < 0.01.
Figure 2. BrdU incorporation (A) and bortezomib sensitivity (B) in
MM cells treated with anti-Notch1 and anti-Notch2. After 2-day
cultivation with 20 μg/ml of control Ig or anti-Notch1 or antiNotch2 antibodies, BrdU incorporation was examined. The relative mean fluorescence intensity (MFI) of the ratio between
the MFI of antibody staining and the MFI of control IgG staining
is shown. After 2-day cultivation with control Ig or antibodies,
the cells were exposed to bortezomib overnight, and the percentage of annexin V+ apoptotic cells was evaluated using FCM.
The data are expressed as mean ± standard deviation of triplicate experiments. *P < 0.05, **P < 0.01.

expression level of not only NOTCH1 but also JAG1 was elevated in the Notch1+ cell fraction compared with that in
Notch1– cells, and the NOTCH2 mRNA expression level did not
differ between the two MM fractions (Fig. 4A). The cell pro­liferative advantage and antiapoptotic responses induced byanti-MM agents in the Notch1+ MM cell fraction was suppressed by anti-JAG1 antibody (Fig. 4B and 4C). NOTCH1 mRNAlevels in Notch1+ and Notch1– MM cell fractions were notaltered by treatment with anti-JAG1 antibody (data notcells (data not shown). These data suggested that Notch1

shown). In BM samples of some MM patients, NOTCH1 mRNA

­molecules may be associated with cell proliferation signalinglevels were higher in CD38+CD138+ plasma cell fractions thanand antiapoptotic responses in MM cells.

in other cell fractions (CD38dimCD138– cells), while the expression pattern of JAG1 mRNA differed in each sample (Fig. 5).

Cell characteristics of Notch1+ and Notch1– U226 cells

Consistent with these results, the mRNA expression of+–antiapoptotic gene p21 (cyclin-dependent kinase inhibitor 1A;

cell fractions without any stimulation (Fig. 1C and Fig. 3A),

CDKN1A) and cyclin genes CCND1 and CCND2 was higher in+

the Notch1+ than in the Notch1– U266 cell fraction (Fig. 6). On­fractions. BrdU incorporation in Notch1 U226 cells was sig­

the other hand, the apoptotic genes Bcl-2-associated X protein

Some MM cell lines contained both Notch1 and Notch1
and we thus evaluated the cell characteristics of Notch1+–

nificantly higher compared with that in Notch1 cells (Fig. 3B).

(BAX), BCL2-associated agonist of cell death (BAD), Fas-associated

The percentage of apoptotic cells induced by melphalan anddeath domain protein (FADD), Caspase-3 (CASP3), CASP8, and+bortezomib was significantly lower in the Notch1 U226 cell–

fraction than in the Notch1 fraction (Fig. 3C). The mRNA
4

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CASP9 were higher in the Notch1– cell fraction (Fig. 6).

Notch1–JAG1 signaling pathway in myeloma

Figure 4. Cell proliferation and drug sensitivity of U266 cells treated with anti-JAG1 antibody. (A) The mRNA expression of NOTCH1, NOTCH2, and
JAG1 was quantified using real-time PCR in Notch1+ and Notch1– U266 cells. BrdU incorporation (B) and bortezomib (BOR) sensitivity (C) in
Notch1+ and Notch1– U266 cells treated with anti-JAG1. After 2-day cultivation with 20 μg/ml of control Ig or anti-JAG1antibody, BrdU incorporation was examined. Relative MFI in the ratio between the MFI of antibody staining and MFI of control IgG staining is shown. After 2-day
cultivation with antibodies, the cells were exposed to bortezomib overnight, and the percentage of annexin V+ apoptotic cells was evaluatedusing FCM. The data are expressed as mean ± standard deviation of triplicate experiments.

Discussion

expression was observed in primary MM cells in BM biopsy
specimens [14]. The blockade of both Notch1 and JAG1

Our results showed that Notch1, but not Notch2, on MM

inhibited the interaction between MM cells and suppressed

cells induces aggressive myeloma behavior including cellaggressive myeloma behaviors including cell proliferative

proliferative potential and antiapoptotic responses against­

potential and drug resistance. When MM cells were co-­anti-MM agents via binding with JAG1.

cultured with JAG1-­expressing 3T3 fibroblasts, activation of

We first demonstrated that NOTCH1 mRNA levels were

the Notch1 pathway protected MM cells from anti-MM agent-­­significantly lower than those of NOTCH2 in MM cell lines andinduced apoptosis via NF-κB activity [12, 15, 16]. Furthermore,primary MM cells, while cell-surface Notch1 expression was

the drug-induced apoptosis and cell proliferation in Notch1-­

detectable. On the other hand, JAG1 mRNA levels were higher

positive MM cell lines, but not in the Notch1-negative cellin comparison with those of other Notch ligands. Other

line, were decreased and increased by JAG1-expressing 3T3

researchers reported that the protein expression of Notch

fibroblasts, respectively [12]. Notch1 molecules transmit­family proteins and their ligands was observed in 8 different­positive signals to MM cells though homotypic and hetero-human MM cell lines, as shown by Western blotting analysis

typic interactions with JAG1-­expressing MM cells and stoma[12]. In immunohistochemical analysis, plasma cells from 92%

cells [10]. Notch activation leads to the transcriptional activa-

and 78% of MM patients expressed Notch1 and JAG1, respec-tion of Notch and Jagged, and the expression levels of these

tively, although cells from patients with monoclonal gammop-

molecules go hand in hand (high Notch and high Jagged)

athy of undetermined significance (MGUS) did not, suggesting[17]. Similarly, both NOTCH1 and JAG1 mRNA levels werethat their expression may be associated with MM evolving

higher in the Notch1+ cell fraction of U266 cells than in thefrom MGUS [13]. Further, Notch1, Notch2, and JAG1 over­Notch1– cell fraction. Furthermore, our study showed that

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ISHIBASHI et al.

Figure 6. Expression of apoptotic and antiapoptotic genes (A) andcell cycle-related genes (B) in Notch1+ U266 cells analyzed using
RT-PCR. Data are represented as fold difference compared withthe data for Notch1– U266 cells.

environment [11]. Therefore Notch1–JAG1 and Notch2–JAG1
interactions might operate as different pathways leading toaggressive tumor characteristics and induce aggressive MM
cell behaviors and MM-associated bone disease, respectively.
Notch receptors are bound by its ligands, and its C-terminal
fragment within the transmembrane domain is cleaved by
γ-secretase, resulting in the release of the Notch intracellulardomain (NICD) fragment [10, 16, 18]. The NICD fragment
released by γ-secretase is translocated into the nucleus andFigure 5. mRNA expression of NOTCH1 and JAG1 in BM cells. (A) Dot
plots showing the percentage of CD38+CD138+ and CD38dim
CD138– cells in BM cells from MM patients. The mRNA expression of NOTCH1 (B) and JAG1 (C) in CD38+CD138+ and CD38dim
CD138– cell fractions.

subsequently induces the transcription of Notch target genes
by interaction with the nuclear transcription factors, i.e.,­protein C protein binding factor 1 (CBF1; also known as RBP-Jκ
or CSL for CBF1/Su[H)/Lag1) and Mastermind-like protein(MAML) [10, 16, 18]. Our data showed that the mRNA expression of cell cycle-related genes CCND1/2 and antiapoptotic

NOTCH1 mRNA expression in plasma cells was higher than ingene p21 in Notch+ MM cells was upregulated in comparison

other BM cells, whereas the expression pattern of JAG1 varied

to that in Notch1– cells. Similarly, the expression of phosphory-

in some MM patient samples. The reason for this is unclear,

lated ERK and AKT, c-Myc, Bcl-2, p21, cyclin D1, and cyclin E

and the differences in JAG1 expression patterns on MM cells

was downregulated in larynx epithelioma cancer and non-or other BM cell components might reflect differences insmall cell lung cancer cell lines by Notch1 knockdown [19, 20].

the BM microenvironment in each MM patient. In actuality,

γ-Secretase inhibitor treatment decreased the expression of

Notch1–JAG1 signaling may be transmitted to MM cells via

Notch-targeted genes in T-cell acute lymphoblastic leukemia

homotypic or heterotypic interactions, as shown in JAG1

cells [21]. When MM cell lines were co-cultured with JAG1-­

expression patterns in the BM cells of individual MM patients.

induced HtTA-jag10 cells pretreated with tetracycline, Notch

On the other hand, we and other researchers have notsignaling in MM cells promoted cell proliferation, and this phe-resolved the question of why Notch1, but not Notch2, signal-

nomenon was suppressed by treatment with the γ-secretaseing is associated with MM cell progression. MM cell-­derived

inhibitor DAPT [14]. In addition, Notch signaling induced inter-

Jagged ligands boost Notch2, but not Notch1, signaling inleukin (IL)-6 production not only in MM cells but also in BM

monocytes and induce mature osteoclasts in the BM micro­

stroma cells, and autocrine and paracrine IL-6 supported MM

6

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Notch1–JAG1 signaling pathway in myeloma

cell growth [22]. Moreover, Notch signaling in BM stroma cells
induced the production of IL-6, vascular endothelial growth
factor, and stromal cell-derived factor alpha (SDF1α; CXCL12)in the BM niche of MM, which play an crucial role in tumor
growth, survival, and migration in MM progression [10, 23–26].
Therefore those studies suggested that Notch–JAG1 signaling
might accelerate MM progression via the interaction with not
only MM but also stromal cells in the BM niche.
In this study, we revealed that JAG1–Notch1 signaling isimportant in myeloma pathogenesis. This underscores thetheory that Notch1–JAG1 may be a potential therapeutic­target in MM patients.

Acknowledgments
This work was supported by the Institute of Medical Sciences, the University of Tokyo (IMSUT) Joint Research Project
FY2017.

Author Contributions
M.I. conceived the study, designed and performed theresearch, analyzed and interpreted data, and wrote and edited
the manuscript. H.T. conceived the study, and designed andedited the manuscript. K.U. performed the research and analyzed and interpreted data. Y.I. discussed the data and gave
conceptual advice. H.T. and K.I. collected patient samples andanalyzed the clinical status of patients. M.R. gave conceptual
advice.

Conflicts of Interest Disclosures
The authors declare no competing financial interest related
to this work.

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