International Journal of Myeloma 3(1): 2–11, 2013
©日本骨髄腫学会

REVIEW

Microenvironment for myeloma growth and drug resistance
Masahiro ABE1
Bone provides a unique microenvironment for myeloma (MM) cell growth and survival, including niches to foster
clonogenic MM cells. MM cells stimulate bone resorption by enhancing osteoclastogenesis, while suppressing boneformation by inhibiting osteoblastic differentiation from bone marrow stromal cells, leading to extensive bone
destruction with rapid loss of bone. MM cells alter the microenvironment through bone destruction in bone where
they colonize, which in turn favors tumor growth and survival, thereby forming a vicious cycle between tumor­progression and bone destruction. MM is still difficult to be cured despite the recent implementation of new agents,
and its bone disease also remains a significant clinical problem. Further elucidation of the molecular mechanisms oftumor-bone interactions and tumor growth in the bone microenvironment will provide us with new approaches
that have a real impact on both bone disease and tumor progression.
Key words:

myeloma niche, Pim-2, Glycolysis, clonogenic myeloma cells, side population

by stromal cells and osteoblasts in the presence of MM cells 8).

1. Introduction

The levels of DKK-1 in sera and bone marrow plasma are
Multiple myeloma (MM) has a unique propensity to expand

increased in MM patients and correlated with the presencealmost exclusively in bone, and develop devastating boneof osteolytic lesions 9). Besides these Wnt inhibitors, several

destruction. MM cells stimulate bone resorption by enhancing

factors derived from MM cells and/or their surrounding­

osteoclastogenesis, and suppress bone formation by inhibiting

microenvironment in bone such as IL-3, IL-7, TNFα, and TGF-β

osteoblastic differentiation from bone marrow stromal cells,

appear to suppress osteoblastic differentiation 1,2,10). More

1,2)

. Cytokines aberrantly

recently, activin A and sclerostin, potent inhibitors of osteo-

over-produced by MM cells, including macrophage inflamma-

blastogensis, have been demonstrated to be over-produced

tory protein (MIP)-1α and MIP-1β as well as receptor activatorin bone lesions in MM 11–13). Therefore, multiple factors actof nuclear factor-κB (RANK) ligand up-regulated in bone

together to eventually develop extensive bone destruction­marrow stromal cells play a major role in the enhancement ofin MM.

leading to extensive bone destruction

3–5). In contrast

Bone provides a unique microenvironment for normal

to enhanced osteoclasto­genesis in MM, osteoblastic differen-

hematopoietic and cancer cell growth, including niches totiation from bone marrow stromal cells is suppressed to cause­foster cancer as well as hematopoietic stem cells. The factextensive bone destruction with rapid loss of bone. Althoughthat MM cells grow and expand almost exclusively in the bone

a canonical Wingless-type (Wnt) signaling pathway plays amarrow suggests the importance of the bone marrow micro-

critical role in osteoblast differentiation, MM cells have beenenvironment in supporting MM cell growth and survival. Whiledemonstrated to secrete soluble inhibitors of the canonicalMM cells perturb a normal bone metabolism to develop bone

Wnt signaling pathway, including Dickkopf-1 (DKK-1) 6) anddestruction, a crosstalk between MM cells and the micro­

osteoclastogenesis and bone resorption in MM

7)secreted Frizzled-related protein (sFRP)-2 . DKK-1 is also secreted

environment in bone lesions leads to the progressive vicious
cycle phase of tumor growth and bone destruction 2,14). In

Received: March 3, 2013, accepted: April 19, 2013
1
Department of Medicine and Bioregulatory Sciences, University ofTokushima Graduate School of Medical Sciences
Corresponding author: Masahiro ABE
Department of Medicine and Bioregulatory Sciences, University ofTokushima Graduate School of Medical Sciences, 3-18-15
Kuramoto-cho, Tokushima, 770-8503, Japan
E-mail: masabe@clin.med.tokushima-u.ac.jp

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International Journal of Myeloma

vol. 3 no. 1 (2013)

addition to the growth advantage that MM cells have acquired
through genetic alterations and clonal selection, the cellular
microenvironment skewed in MM endows MM cells with anadditional growth and survival potential. In this review, current
understanding of the mechanisms of MM progression anddrug resistance in bone lesions and therapeutic approaches
based on these mechanisms will be discussed.

Bone formation
Matureosteoblast

Myelomacell

Growth/Survival

mote MM cell homing or colonization in the bone marrow,
which up-regulates the production by stromal/pre-osteoblastic
cells of growth and anti-apoptotic factors and CAM-DR for MM
cells. In contrast to the above factors over-produced in the bone

Myeloma nichemarrow in MM, a murine model permissive for MM growthdifferentiation

revealed decreased levels of host-derived adiponectin 18).

StromalcellOCosteoclast

Endothelial
cellInterstingly, adiponectin appears to be decreased in the serum

Angiogenesis

Adiponectin was found to increase MM cell apoptosis throughBone resorptionof patients with MGUS that subsequently progressed to MM.
activation of the AMP kinase pathway, and tumor burden andbone disease were enhanced in MM models using adiponectin-

Figure 1. MM niche in bone. MM
cells 1stimulate bone resorption by
Figureenhancing osteoclastogenesis, while suppressing bone formation by inhibiting osteoblastic differentiation from bone marrowstromal cells, leading to extensive bone destruction with rapid
loss of bone. MM-induced cell types in MM bone lesions, namely
osteoclasts, vascular endothelial cells, and bone marrow stromalcells with defective osteoblastic differentiation create suitablefor MM growth and survival and confer a drug resistance to MM
cells, which can be called an “MM niche”.

deficient mice.
2.2. OCs
Other than stromal cells, OCs induced by MM cells are also
among the major cellular components of the bone marrow
microenvironment. Intriguingly, OCs enhance MM cell growthand survival via an interaction between MM cells and OCs 19,20).
The effects of OCs on MM cell growth seem more potent
than those of primary bone marrow stromal cells, while MM

2. MM nichecells more firmly adhere to stromal cells and confer drug
­resistance 20). Similar to bone marrow stromal cells, OCs protect

By analogy with the formation of a cancer pre-metastatic

MM cells from apoptosis induced by anti-cancer drugs, includ-niche by the migration and seeding of hemopoietic cells to

ing doxorubicin. BAFF and a proliferation-inducing ligand

metastatic sites, MM cell-induced cell types in MM bone

(APRIL), members of the tumor necrosis factor (TNF) family,

lesions, namely osteoclasts (OCs), vascular endothelial cells,

have been implicated as survival factors for ­myeloma cells 21,22).

and bone marrow stromal cells with defective osteoblastic

OCs are a predominant cell type producing these factors in the­differentiation, appear to play an important role in creating abone marrow in MM 23). Treatment with TACI-Fc, a decoy recep-

beneficial cellular environment for MM cell growth and sur-tor for both BAFF and APRIL, markedly enhanced apoptosis ofvival as a feeder for MM cells. Such a microenvironment can

MM cells in the presence of OCs, indicating a supporting role

be construed as the “MM niche” (Figure 1).

for BAFF and APRIL in OC-mediated MM cell growth and survival 24,25). Ge et al. reported that the serine protease fibroblast­

2.1. Bone marrow stromal cells

activation protein (FAP) is up-regulated in OCs after coculture

Among cell components in the bone marrow, the roles ofwith MM cells 26). Knockdown of FAP expression reduced­MMbone marrow stromal cells in MM cell growth and survival

cell survival in co­cultures. In addition, FAP is also expressed inhave been most extensively studied. The interaction between

osteogenic cells, bone marrow stromal cells, adipocytes, andMM cells and stromal cells confers MM cell homing, growth,

vascular e­ ndothelial cells in the bone marrow.

survival, and resistance to chemotherapy 15). MM cells stimulate

Hexokinase II (HKII), a key enzyme of glycolysis, is widely

stromal cells to produce various growth and anti-apoptotic

over-expressed in cancer cells 27) (Figure 2). HKII has beenfactors for MM cells, including IL-6, IGF-1, SDF-1α, IL-21, B-cell-demonstrated to be over-expressed in most MM cells 28). The

activating factor (BAFF), and VEGF, while inducing RANK ligand

inhibitor of HKII 3-bromopyruvate (3BrPA) promptly and sub-to enhance osteoclastogenesis. Importantly, the adhesion of

stantially suppresses ATP production and induces cell death inMM cells to stromal cells and their extracellular matrices (ECM)

MM cells 29). Interestingly, cocultures with OCs but not bone

via VLA-4 or VLA-5 confers cell adhesion-mediated drug resis-

marrow stromal cells enhanced the phosphorylation of Akt

16)tance (CAM-DR) on MM cells . Autocrine activation of VLA-4

along with an increase in HKII levels and lactate production in

on MM cells by MM cell-derived MIP-1 and up-regulation of

MM cells. The PI3K-Akt signaling has been demonstrated to

MIP-1 production by MM cells through VLA-4-VCAM-1 interac-phosphorylate HKII to facilitate its association with a mitochon-tions appear to form a positive feedback loop between thedrial membrane, and thereby stabilize HKII to post-translationally17)

adhesion of MM cells and their MIP-1 production . In addi-

increase intracellular HKII levels 30). Consistently, the enhance-tion to osteoclastogenesis, MIP-1 has been suggested to pro-ment of HKII levels and lactate production in MM cells by OCs

International Journal of Myeloma

vol. 3 no. 1 (2013)3

DrugsGlucoseduction of osteoclastogenic activity by vascular endothelial
cells 35). Osteopontin is subject to enzymatic cleavage, and its

ABCtransporters

Glutfragments become functionally active 36). OCs also produce
matrix metalloproteinase (MMP)-9, which has been demon-ATPstrated to be responsible for angiogenesis induced by OCs 37).

ADP + Pi

MMP-9 may affect the activity of other factors elaborated inglycolysis

bone lesions in MM including osteopontin. Therefore, a close
link between MM cells, OCs, and vascular endothelial cells can

Biosynthesis

be established in MM bone lesions, thereby forming a vicious
cycle involving bone destruction, angiogenesis, and MM

Growth / survival

expansion (Figure 1).

Figure 2. Aerobic glycolysis Figure(the Warburg
effect). Malignant cells
2
increase their expression of glycolytic enzymes and glucose
uptake to markedly enhance glycolysis (aerobic glycolysis; theWarburg effect), which leads to the production of a large
amount of ATP and biomass such as nucleic acids and lipids
essential for cell survival and division. ABC transporters areaberrantly over-expressed in drug-resistant cancer cells andside population (SP) cells. ABC transporter function in thesecells appears to be mediated by ATP abundantly produced
through enhanced glycolysis. The inhibition of glycolysis canabolish ATP production as well as biomass synthesis in cancer
cells without compromising ATP production and cell metabolism
in normal quiescent cells; and thus enhanced glycolysis may
become a novel cancer-specific target for anti-cancer treatment.

2.4. Niches for MM cancer stem cell (CSC)-like cells
The presence of CSCs or cancer-initiating cells have been
demonstrated in various types of cancers, and regarded as apredominant cause of drug resistance. Likewise, MM CSCs
have been postulated, and are considered to contribute todisease­relapse through their drug-resistant nature 38,39). Thus,
MM CSCs become among the most important targets in thetreatment for MM. However, MM CSCs are still conceptual inmany senses, because of the lack of sensitive and reliable
methods to identify them. Besides clonotypic B cells, phenotypically distinct MM plasma cell fractions have been demonstrated to possess a clonogenic capacity 40–42), leading to longlasting controversies regarding the cells of origin in MM orwere mostly abrogated by the PI3K inhibitor LY294002, sug-

MM-initiating cells. It is hard to explain the clonogenic capacity

gesting activation of glycolysis in MM cells by OCs via the PI3K-

of differentiated MM cells only with the notion of a one-way

Akt-HKII pathway. Treatment with 3BrPA was able to markedly

hierarchical model from MM CSCs to differentiated MM cells.

induce cell death in MM cells even in cocultures with OCs.

MM CSCs may not be a static population but survive as

These observations collectively suggest the contribution ofphenotypically­and functionally different cell types via theOCs to the aggressiveness or drug resistance of MM cells. Thus,

transition between stem-like and non-stem-like states in local

MM cells and OCs directly interact to augment their growth

microenvironments, as observed in other types of cancers.

and activity, thereby forming a vicious cycle that leads to

Recently, Chaidos et al. elegantly addressed a phenotypic

extensive bone destruction and MM expansion (Figure 1).

and functional interconvertible state between CD138+ andThese observations provide a rationale for therapeutic strate-

CD138− cells 43). They identified a distinct subpopulation withgies targeting the OC-MM interaction.

CD19− CD38++ CD319+ CD138− immunophenotype in clonotypiccells in both the bone marrow and circulation in patients with

2.3. Angiogenesis

MM, and termed as pre-plasma cell. They identified a CD138+

Similar to osteoclastogenesis, angiogenesis is enhanced inplasma cell to pre-plasma cell transition within clonotypic

the bone marrow in MM in parallel with tumor progres-subpopulations in the bone marrow samples of 30 patientssion 31,32). Bone marrow stromal cells as well as MM cells secretewith MM by dynamic mathematical models. Together with theangiogenic factors including vascular endothelial growthobservations with the forward transition of clonotypic CD19+

33,34).

cells and/or CD138low cells to differentiated CD138+ MM plasma

In vascular endothelial cells, the VEGF receptor VEGF-R2 mutu-

cells 38,39,44), bidirectional transitions between differentiatedally interacts with αVβ3 integrin after the binding of theirand undifferentiated MM clonotypic cells are suggested in

respective ligands to efficiently transduce their downstream

patients with MM (Figure 3). Because the inoculation of

signaling and induce angiogeneisis. Of note, OCs constitutively

phenotypically­differentiated MM cells alone could form

secrete a large amount of the proangiogenic factor osteopontin,

MM in xenograft models, which is transplantable to secondary

a ligand of αVβ3 integrin, which cooperates with VEGF secreted

and tertiary recipients 40,41), MM-initiating cells seem to arise

from MM cells to enhance angiogenesis and induce the pro-from MM cell population in vivo. Collectively, these findings­factor (VEGF) and basic fibroblast growth factor (bFGF)

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International Journal of Myeloma

vol. 3 no. 1 (2013)

(A)Circulation

Circulation(B)

Bone marrow niche

Extra-osseous niche

self-renewal
MM CSC

self-renewal
MM CSC

Transition?
clonotypic
B cellTransition?

MM cellclonotypic
B cell

Pre-PC

Pre-PC

MM cellFigure 3. Localization of clonotypic
cells
cells. A.3Bone marrow­(BM) niche for
Figure3 in MM and transitions between MM progenitors and differentiatedFigureMM cells and their progenitors. MM cells are phenotypically and functionally heterogeneous populations. Clonotypic B cells have been
demonstrated to be able to give rise to monoclonal immunoglobulin-secreting plasma cells. Besides clonotypic B cells, phenotypicallydistinct MM plasma cell fractions have been demonstrated to possess a clonogenic capacity. A distinct subpopulation termed as a preplasma cell (pre-PC) with CD19− CD38++ CD319+ CD138− immunophenotype in clonotypic cells is identified in both the bone marrow andcirculation­of patients with MM. Bidirectional transitions between pre-PC and differentiated MM cells have been demonstrated. These
clonotypic cells egress from the bone marrow into circulation and home back to the bone marrow. The existence of phenotypic as well asfunctional plasticity between MM cancer stem cells (CSCs) and non-CSC populations is also suggested. B. Extra-osseous niches for MM
progenitors. When human CD138+ mature MM cells were injected via tail veins in human MM-permissive mice, the spleen and liver were
found to contain predominantly immature clonotypic plasma cells, including pre-PCs, rather than CD138 + mature MM cells, although CD138+
mature MM cells composed the majority of clonotypic cells in the bone marrow. Thus, extra-osseous tissues are thought to harbor rather
immature clonotypic cells egressed from the bone marrow, and provide niches for clonogenic MM cells to foster them. Immature clonotypiccells or clonogenic MM cells colonized in extra-osseous may egress again and home to the bone marrow.

imply the existence of phenotypic as well as functional plas­for MM-initiating cells to cause relapse. Taken together, theseticity between CSCs and non-CSC populations in MM cells.

results suggest dedifferentiation of mature MM cells in vivo

As reported for SCID-hu or SCID-rab models in which humanand preferential localization of immature MM clonotypic cells

MM cells were injected into bone marrow cavities of subcuta-or MM progenitors in certain extra-osseous microenviron-neously implanted human or rabbit bones, MM growth wasments as well as the bone marrow. Further studies are

restricted to the implanted bones, suggesting that human orneeded to explore the localization of MM CSC-like cells andrabbit bones serve niches for MM CSCs in these experimental

the mechanisms of their tumorigenicity and plasticity uponmodels 40,45). Furthermore, Yaccoby demonstrated that ininteraction with their preferential microenvironment.

cocultures with osteoclasts, differentiated MM cells became
plasmablastic along with reduced expression of CD38 and3. Targeting MM cell-MM niche interaction

CD138, and developed resistance to dexamethasone-induced
apoptosis 46). Interestingly, Chaidos et al. found that when+

MM remains incurable even with the implementation ofhuman CD138 mature MM cells were injected into immuno-

novel anti-MM agents, high-dose chemotherapies, anddeficiency mice via tail veins, the spleen and liver contained

immunotherapies, and thus there is a need to target and−disrupt the MM niche to improve the efficacy of the present

CD38++ CD319+ CD138− pre-plasma cells and CD138low plasma

therapeutic modalities against MM progression as well as MM

predominantly immature clonotypic­cells, namely CD19++

cells, rather than CD138 MM cells, although CD138 maturebone disease.

MM cells composed the majority of clonotypic cells in thebone marrow 43) (Figure 3). Furthermore, clonotypic B cells

3.1. Inhibition of osteoclastogenesis

appear to play a role in disease progression or recurrence in

The administration of inhibitors of OC activity, including

MM. The frequency of circulating clonotypic B cells have beenbisphosphonates, RANK-Fc, and osteoprotegerin, not only

demonstrated to increase in parallel­with disease progres-­prevents MM-induced bone destruction but also interferes

43)

sion . Clonotypic B cells are detected not only at diagnosis orwith tumor progression in animal models of MM in someat relapse but also persist even in patients with MM achieving

reports 47–49). The repeated administration of bisphosphonatesa molecular remission in bone marrow samples after treat-

has also been reported to reduce the tumor burden withoutments 43). Therefore, clonotypic B cells appear to be a reservoir

chemotherapy in a portion of MM patients 50,51). These observa-International Journal of Myeloma

vol. 3 no. 1 (2013)5

tions are consistent with the notion that an interaction

matrices through enhanced bone resorption and activated by

between OCs and MM cells may play an important role in MM

acids and MMPs secreted from OCs. We have demonstrated

expansion in the bone marrow. In addition, the MM-OC inter-that blockade of TGF-β action releases stromal cells from theiractions create an acidic milieu by protons produced by OCs

differentiation arrest by MM, and that terminally differentiatedand lactate by proliferating MM cells (the Warburg effect),osteoblasts inhibit MM cell growth and survival and potentiate

which blunts cytotoxic effects of various chemotherapeutic

responsiveness to anti-MM agents 10). These results support theagents as well as the activity of cytotoxic immune cells.

idea that the suppression of osteoblastic differentiation by MM

Therefore, potent anti-resorptive therapy together with tumor

both accelerates bone loss and creates an MM niche to provide

reduction is needed to effectively suppress osteoclastogenesis,

favorable conditions for MM growth and survival (Figure 1).

thereby disrupting the vicious cycle between osteoclasto­

Thus, the induction of osteoblastic differentiation throughgenesis and MM expansion.

TGF-β inhibition may provide a novel approach to ameliorat-

OCs produce the anti-apoptotic factors BAFF and APRIL, as

ing bone destruction and tumor progression in MM.

noted above. BAFF is also produced by bone marrow stromal

Activin A is a TGF-β superfamily member associated with acells 52). Gene expression of TACI, a receptor for these factors,

variety of fundamental biological processes, including embryo-in MM cells can distinguish tumors with a signature of micro-

genesis, organ development, gonadal hormone signaling, andenvironment dependence 23). To target these factors, TACI-Ig

erythropoiesis. It also affects bone remodeling, and plays a(atacicept) and anti-BAFF neutralizing antibody have been

crucial role in the skeleton 68). It has been reported that bonedeveloped and shown to exert anti-MM effects in preclinicalmarrow plasma levels of activin A are increased in MM patients. Denosumab, a fully human monoclonalwith bone lesions 11). MM cells induce the release of activin A

antibody against RANK ligand that inhibits the RANK ligand-from bone marrow stromal cells in part via activation of their

RANK system, is under worldwide clinical trials with patientsJNK pathway 11) (Figure 1). Furthermore, activin A inhibitswith MM.

osteoblastic differentiation and bone formation by stimulating

animal models

25)

intracellular signaling protein Smad2 activity and inhibiting
3.2. Induction of bone formation

distal-less homeobox (DLX)–5 expression. The soluble activin

A reverse correlation between osteoblastic differentiationand MM tumor growth has been reported in patients with MM
53–55)

type IIA (ActRIIA)-Fc fusion protein is now under clinical development for patients with MM.

andA large amount of ECM proteins is accumulated withcarfilzomib 56). Serum levels of bone-specific alkaline phospha-

osteoblastic differentiation. TGF-β-binding small leucine-richtase were found to be elevated after treatment with bortezo-

proteoglycans, biglycan and decorin, were found to be among

mib, which were inversely correlated with a reduction in thethe most up-regulated proteins produced by terminally dif-treated with the proteasome inhibitor bortezomib. Such bone anabolic effects of bortezomib

ferentiated osteoblasts 10). These proteoglycans bind various

and their correlation with tumor regression were further

growth factors and modulate their actions. Decorin hastumor burden

57,58)

59). MM growth inhibi-

been identified among osteoblast-derived factors responsibletion associated with osteoblastic differentiation has also been

for the suppression of MM cell growth and survival 69). Modifi-

observed in MM animal models treated with other anabolic

cation of the sulfation status of extracellular proteoglycan

agents such as anti-DKK1 antibody 60,61), lithium chloride 62),

chains markedly regulated the binding and signal transduc-demonstrated­in MM animal models

12,63), as well as with enforced expression

tion of a number of growth factors and cytokines. Forcedof Wnt3a within bone 64). These observations suggest that

expression of extracellular heparan sulfate 6-O-endosulfatases,

anti-MM activity emerges with osteoblastic differentiation, andwhich are down-regulated in MM cells, was reported tothat MM cells may protect themselves from such osteoblast-markedly promote the deposition of ECM within the tumor

mediated growth suppression by inhibiting the terminal dif-

microenvironment, along with a marked reduction in MM

ferentiation of osteoblasts (Figure 1). The pro-anabolic agent

tumor growth in vivo 70). These findings suggest that modi­

BHQ880, an anti-DKK-1 monoclonal antibody, is currently being

fication of the sulfation status of proteoglycans can have aevaluated in clinical trials to test the promising preclinicalconsiderable impact on MM cell growth and survival. A

and activin A inhibitor

65)

results .

variety­of ECM proteins and enzymes affecting the sulfation

Transforming growth factor-β (TGF-β) is a potent inhibitor

status, including sulfatases and sulfotransferases, have been

66).

found to be targets of Runx-2, an essential osteogenic

It is produced by osteoblasts and osteocytes, and depositedtranscription factor, and up-regulated in mature osteo-of terminal osteoblastic differentiation and mineralization67)

in bone matrices as its latent form . In MM bone destructive
lesions, TGF-β appears to be abundantly released from bone
6

International Journal of Myeloma

vol. 3 no. 1 (2013)

blasts 71).

3.3. Targeting MM cell signaling pathways up-regulated

MM cells to chemotherapeutic agents by Pim inhibition war-in bone lesions

rants further study on combinatory treatment of anti-MM

A number of intracellular signaling mediators, including

agents such as bortezomib and melphalan with Pim inhibitors.

NF-κB, Akt, p38MAPK, and the adaptor protein p62, have been

Inhibition of the IGF-1/PI3K/Akt pathway by Akt inhibitors has

well demonstrated to be activated in both MM cells and theirdrawn considerable attention as a new therapeutic modality

surrounding environmental cells and regulate the pathologicalagainst MM 78,79). However, because the survival pathways

bone metabolism and tumor growth in MM.

elicited by Pim kinases seem independent of the PI3K/Akt

In pursuing factors responsible for MM tumor survival inpathway 72,80), Pim kinases should be targeted together tobone lesions, we have found that the serine/threonine kinase

improve the anti-MM efficacy of PI3K/Akt pathway inhibitors.

Pim-2 is constitutively over-expressed as a critical pro-survival

As Pim-2 expression is not increased in normal bystander cells,

mediator in MM cells, and its expression is further up-regulated

Pim-2 seems to be a specific target for the treatment of MM.

in cocultures with bone marrow stromal cells or OCs 72) (Figure

Specific inhibitors for Pim-2 are under development at several

4). Pim-2 has been identified using cDNA microarrays as one ofpharmaceutical companies as anti-cancer agents 76), and may

the 34 most highly over-expressed genes in MM cell lines inbe available in the near future.

73)

comparison with lymphoma and AML cell lines . Although
Pim-2 appears to be the most up-regulated member of the
Pim family, MM cells also expressed Pim-1, but rarely Pim-3,

3.4. Targeting glycolysis in drug-resistant and clonogenic
MM cells

at various levels. Pim-1 and Pim-2 were first identified as

The most important property of CSCs is that they are resis-

oncogenic factors based on observations of the development

tant to chemotherapeutic agents. Targeting MM CSCs is clini-of lymphomas in transgenic mice over-expressing thesecally relevant, and various approaches have been proposed to74,75). In addition to enhancing oncogenic and survival

target molecular, metabolic and epigenetic signatures andsignals, Pim kinases have also been shown to be involved in

the self-renewal signaling pathway characteristic of MM CSC-

kinases

the expression and activation of drug efflux transporters

76).

like cells.

Pim-1 phosphorylates the ATP-binding cassette (ABC) trans-

The side population (SP) phenotype is characteristic of stem

porter ABCG2, also known as breast cancer resistance protein

cells in various normal tissues 81). CSCs also exhibit high levels(BCRP), and thereby promotes its multimerization with itsof ABC transporter activity and confer drug resistance besides

77)

stable membrane expression, leading to drug resistance .

their clonogenic or tumor-initiating capacity. SP cells are

Because ABCG2 is preferentially expressed in MM cells, theidentified by their ability to efflux Hoechst33342 dye, aup-regulation of Pim kinases may involve a drug efflux in MM

substrate for ABCG2, suggesting that SP cells have high ABC

cells to reduce the efficacy of drug treatment. Sensitization oftransporter activity. SP cells have been also demonstrated asa drug resistant fraction in many cancers, and are considered
to contain their CSCs 81–85). In MM, the distinct fraction of SPcells has been detected in both MM cell lines and primary

Myeloma cell
NF-kB
JAK2/
STAT3

MM cells 86).

BAFF
IL-6 APRIL

Pim-2
VCAM-1

VLA-4

Malignant cells increase their expression of glycolytic
enzymes and glucose uptake to markedly enhance glycolysis(aerobic glycolysis; the Warburg effect), which leads to the
production of a large amount of ATP and biomass such as

OCnucleic acids and lipids essential for cell survival and divi-OC

production by oxidative phospohorylation in the tricarboxylic

stromaFigure4 in MM cells in bone lesions.
Figure 4. Enhanced expressionof Pim-2
Pim-2 is a novel pro-survival mediator for MM cells. MM bonemarrow microenvironment up-regulates Pim-2 expression in
MM cells through activation of the JAK2/STAT3 pathway for IL-6
and the NF-κB pathway for TNF family cytokines, TNFα, BAFF
and APRIL, to promote MM cell survival. Pim-2 over-expressedin MM cells in the MM bone marrow microenvironment appears
to be an important therapeutic target.

sion 28,87–89) (Figure 2). In parallel with enhanced glycolysis, ATPacid (TCA) cycle in mitochondria is suppressed through oncogenic alterations including the mutation of p53. Glycolysis in
MM cells has been demonstrated to be further up-regulated in
cocultures with OCs 29). Because ABC transporter activity isdependent on ATP 90,91), and because ATP production in cancer
cells is largely dependent on enhanced glycolysis, inhibition of
glycolysis by 3BrPA was found to promptly and effectively
suppress ATP production and ABC transporter activity in ABC
transporter-expressing malignant cells and restored theirInternational Journal of Myeloma

vol. 3 no. 1 (2013)7

susceptibility to anti-cancer drugs 29). Although the relation-

Acknowledgments

ship between ABCG2 expression and SP phenotype has been
established, other ABC transporters are also involved in theThis work was supported in part by Grants-in-aid for Scientific

SP phenotype with high ABC transporter activity and drug

Research (C) to M.A., and a National Cancer Center Research

resistance. Therefore, the efficacy of strategies targeting aand Development Fund (21-8-5) to M.A. from the Ministry of

single transporter appears to be limited. In contrast, inhibition

Health, Labor and Welfare of Japan. The funders had no role inof glycolysis is able to simultaneously inactivate all types ofstudy design, data collection and analysis, decision to publish,

ABC transporters in cancer cells including SP cells, because

or preparation of the manuscript.

each transporter is dependent on ATP generated largely
through enhanced glycolysis. The SP cells exhibited theConflicts of interest disclosures: The authors declare no com-increased expression of genes involved in the glycolytic path-

peting financial interests related to this work.

way including GLUT1, GLUT3, PDK1 and PFK2; and generated

Referencesa larger amount of ATP and lactate per cell compared to MP
29)cells . Thus, glycolysis appears to be highly accelerated in SP
cells. Furthermore, the ability of RPMI8226 cells to form colonies was completely abolished by relatively low concentrationsof 3BrPA. From these results, it is plausible to assume that
inhibition of glycolysis can disrupt their clonogenic capacityand drug resistant nature in SP cells.

4. Perspectives
MM cells and their surrounding cells in the bone marrow
microenvironment closely interplay with each other to createan MM niche in the bone marrow to promote MM cell growthand protect MM cells from spontaneous and drug-induced
apoptosis (Figure 1). In addition to cytotoxic agents for MM
cells, novel agents targeting bone disease appear to be promising therapeutic strategies for the treatment of MM tumor
expansion. However, because MM remains incurable evenafter effectively debulking the tumor mass, the idea of MM
CSCs has been proposed to represent functional properties ofsmall numbers of cells that have innate resistance to chemotherapeutic agents and the ability to live long and regrow in
local microenvironments. Given the interconversion between
MM CSC-like cells or undifferentiated clonotypic cells andMM cells, we should target all types of clonotypic cells from
MM CSCs to mature MM cells. Although MM cells preferentiallygrow within the bone marrow, we also need to clarify where
MM CSCs localize and how they self-renew upon interaction
with MM micro­environment. The establishment of sophisticated, refined and humanized methods to recapitulate MM
microenvironment, which supports MM CSC-like cell survival
and self-renewal, is urgently needed. Further elucidation of themolecular mechanisms that keep MM clonogenesity and drivethe ­transition between stem-like and non-stem-like states inlocal microenvironments will provide us with new approacheswith crucial impact on the therapeutic paradigm in MM.

8International Journal of Myeloma

vol. 3 no. 1 (2013)

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