TITLE PAGE - Clinical Cancer Research · 2014. 1. 31. · Hematology Program (RD12/0036/0058),...

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TITLE PAGE

Title: Preclinical activity of the oral proteasome inhibitor MLN9708 in myeloma bone disease

Authors and Affiliations:

Antonio Garcia-Gomez1,2,3, Dalia Quwaider1, Miriam Canavese4, Enrique M Ocio1,3, Ze Tian5,

Juan F Blanco3, Allison J Berger6, Carlos Ortiz-de-Solorzano7, Teresa Hernández-Iglesias1,

Anton CM Martens8,9, Richard WJ Groen8, Joaquín Mateo-Urdiales7, Susana Fraile1, Miguel

Galarraga7, Dharminder Chauhan5, Jesús F San Miguel1,2,3, Noopur Raje4 and Mercedes

Garayoa1,2,3

1Centro de Investigación del Cáncer, IBMCC (Universidad de Salamanca-CSIC), Salamanca,

Spain; 2Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León,

Salamanca, Spain; 3Hospital Universitario de Salamanca-IBSAL, Salamanca, Spain; 4MGH

Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;

5Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 6Millennium

Pharmaceuticals, Inc., Cambridge, MA, USA; 7Laboratorio de Imagen del Cáncer, Centro de

Investigación Médica Aplicada, Universidad de Navarra, Pamplona, Spain; 8Department of

Cell Biology and 9Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

Running title: Preclinical activity of MLN9708 in myeloma bone disease

Keywords: MLN9708; proteasome inhibitor; myeloma bone disease.

Financial support:

This work was supported by grants from the Spanish MICINN - ISCIII (PI081825; PI1202591),

Centro en Red of Regenerative Medicine and Celular Therapy from Castilla y León, RTICC

Hematology Program (RD12/0036/0058), Spanish FIS (PS09/01897), MINECO DPI2012-

38090-C03-02 and CIMA (The “UTE project CIMA”). AG-G was supported by the “Spanish

Society of Hematology and Hemotherapy”.

Research. on December 18, 2020. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 31, 2014; DOI: 10.1158/1078-0432.CCR-13-1657

http://clincancerres.aacrjournals.org/

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Corresponding author:

Antonio García; Centro de Investigación del Cáncer, IBMCC (Universidad de Salamanca-CSIC);

Campus Miguel de Unamuno, Avda. Universidad de Coimbra s/n. 37007 Salamanca, Spain. E-

mail: [email protected]. Phone: 34 923 294 812. Fax: 34 923 294 743

Disclosure of potential conflicts of interest: JF San Miguel and N Raje declare consultancy

for Millennium Pharmaceuticals, Inc. A Berger is an employee of Millennium Pharmaceuticals,

Inc. All other authors disclose no potential conflicts of interest.

Other manuscript notes:

● word count (excluding acknowledgements, references and figure legends): 4958

● total number of figures and tables: 6

● no Supplementary Data are uploaded

MAIN TEXT

Abstract

Purpose: MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238

(ixazomib), is a next generation proteasome inhibitor (PI) with demonstrated preclinical and

clinical anti-myeloma activity, but yet with an unknown effect on myeloma-associated bone

disease. Here, we investigated its bone anabolic and anti-resorptive effects in the myeloma

setting and in comparison to bortezomib in preclinical models.

Experimental design: The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast

precursors from healthy donors and myeloma patients, and on osteoprogenitors derived from

bone marrow (BM) mesenchymal stem cells also from both origins. We used an in vivo model of

BM disseminated human myeloma to evaluate MLN2238 anti-myeloma and bone activities.

Results: Clinically achievable concentrations of MLN2238 markedly inhibited in vitro

osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL-

induced NF-κB activation, F-actin ring disruption and diminished expression of αVβ3 integrin. A




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similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast

activity (even in osteoprogenitors from myeloma patients), partly mediated by activation of

TCF/β-catenin signaling and upregulation of the IRE-1 component of the unfolded protein

response. In a mouse model of BM disseminated human MM, orally administered MLN2238

was equally effective as bortezomib to control tumor burden and also provided a marked benefit

in associated bone disease (sustained by both bone-anabolic and anti-catabolic activities).

Conclusion: Given favorable data on pharmacologic properties and emerging clinical safety

profile of MLN9708, it is conceivable that this PI may achieve bone beneficial effects in addition

to its anti-myeloma activity in myeloma patients.

Translational relevance

Second-generation proteasome inhibitors (PI) are now being evaluated as putative alternatives

to bortezomib for the treatment of multiple myeloma patients. In this context, MLN9708,

hydrolyzing to pharmacologically active MLN2238 (ixazomib), is an orally bioavailable PI with

demonstrated preclinical and clinical activity against multiple myeloma, but yet with unknown

beneficial effect on myeloma-associated bone disease (MBD).

Our in vitro data demonstrate that clinically relevant concentrations of MLN2238

promote OB differentiation and function and inhibit OC formation and resorption. In a mouse

model of disseminated human myeloma, oral administration of MLN2238 was found to be at

least equally effective as intraperitoneally administered bortezomib in the control of myeloma

growth and in prevention of bone loss, sustained by both bone-forming and anti-catabolic

activities. This data provide the rationale for clinical assessment of MLN9708´s bone effects in

patients with MBD.

Introduction

Multiple myeloma (MM) is a hematological malignancy resulting from the growth of plasma cells

within the bone marrow (BM) (1). The presence of osteolytic lesions is a hallmark of the

disease, characterized by an increase in bone-resorptive activity and number of osteoclasts

(OCs) and impairment of bone-forming activity and differentiation of osteoblasts (OBs) (2, 3).




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OC hyperactivation and enhanced differentiation from precursors is primarily mediated through

increased secretion of OC-activating factors both by MM cells and other cells from the BM

microenvironment [e.g. receptor activator of NF-κB ligand (RANKL), CCL3, IL7, IL3, activin A],

whereas levels of osteoprotegerin (OPG), a soluble decoy receptor for RANKL, are reduced. At

the same time, OCs promote MM cell survival and growth by the release of IL-6, CCL3, B-cell

activating factor (BAFF) and a proliferation-inducing ligand (APRIL), creating a vicious cycle

between bone lesions and tumour expansion (4, 5). On the other hand, reduced OB formation

and activity is in part sustained through augmented expression of Wnt and BMP signaling

antagonists and other OB suppressive factors [such as DKK-1, soluble Frizzled related protein-

2, sclerostin, transforming growth factor-β, hepatocyte growth factor, activin A, IL7, CCL3 and

tumor necrosis factor-α) [reviewed in (2, 3, 5)], and by blockade of activity of the critical OB

transcription factor Runx2/Cbfa1 via cell-cell contact interactions of myeloma and

osteoprogenitor cells (6).

The incorporation of the first in class proteasome inhibitor (PI), bortezomib, to the anti-

myeloma armamentarium can be considered one of the major milestones in the treatment of

myeloma patients, greatly improving the response rates and overall survival in both front-line

and relapsed/refractory settings (7). Bortezomib is a dipeptide boronic acid, primarily inhibiting

the chymotrypsin-like activity (β5 subunit) of the 20S proteasome with slowly reversible kinetics

(110 min in an in vitro enzyme assay) (8). Myeloma plasma cells seem to be especially sensitive

to bortezomib, and multiple effects have been reported after bortezomib treatment (e. g.

blockade of NF-κB activity, cell cycle arrest, intrinsic and extrinsic induction of apoptosis,

inhibition of DNA repair enzymes, inhibition of adhesion to BM stromal cells), which likely

contribute to the clinical success of this agent in MM [reviewed in (9)]. Moreover, bortezomib

also restrains the progression of myeloma bone disease (MBD), as evidenced by changes in

bone turnover markers and radiologic data favouring bone healing (10-12). This beneficial

impact of bortezomib on bone metabolism is not merely secondary to its anti-myeloma activity,

but rather this agent acts directly both interfering with osteoclastogenesis and resorption and

promoting OB formation and function, as shown in numerous in vitro studies (13-15), and in

myelomatous and non-myelomatous in vivo models (16, 17).




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However, some patients are not sensitive to bortezomib, and main limitations

associated with its use are the eventual development of resistance and the emergence of

peripheral neuropathy (8). This has prompted the development of structurally different second-

generation PIs, with the aim of retaining or improving bortezomib efficacy, overcoming

bortezomib resistance and having a safer toxicity profile. In fact, several next-generation PIs are

currently under various stages of clinical evaluation (7).

In this context, MLN9708 was selected from a pool of boron-containing PIs because of

its oral bioavailability and its distinct physicochemical profile compared to that of bortezomib (8,

18). MLN9708 hydrolyzes to the biologically active compound MLN2238, which preferentially

and reversibly inhibits the β5 chymotryptic-like subunit of the proteasome (IC50 = 3.4 nM), albeit

with a substantially shorter dissociation half-life than bortezomib (18 min in an in vitro enzyme

assay) (8). This differential binding kinetics is thought to play an important role in the tissue

distribution of MLN2238, with a stronger and more sustained proteasome inhibition in xenograft

tumors, and a weaker and less sustained effect in whole blood, as compared to bortezomib

(18). MLN2238 has shown in vitro cytotoxicity on MM cells, even on those resistant to

conventional therapies (including bortezomib), and also inhibited tumor growth and reduced

recurrence in a human plasmacytoma model (19). In fact, clinical trials of orally administered

MLN9708 (now entering phase III) have evidenced its tolerability with infrequent peripheral

neuropathy (20, 21) and clinical anti-myeloma activity either alone or in combination with other

anti-myeloma drugs (22). Signs of additional beneficial bone effects of MLN2238 have been

observed in a model of disseminated mouse plasma cell malignancy (23), suggesting that this

agent, similar to bortezomib, may also display direct regulatory effects on bone remodeling. We

have therefore conducted in vitro studies to comparatively evaluate the ability of MLN2238

(relative to that of bortezomib) to promote the osteogenic differentiation from mesenchymal

progenitors from both healthy donors and myeloma patients, and also to inhibit OC formation

and function. In addition, we present evidence of anti-myeloma and global bone anabolic effects

of orally administered MLN2238 in a BM-disseminated human myeloma mouse model,

demonstrating both anti-resorptive and bone-anabolic activities of this agent.

Materials and methods




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Reagents and immunochemicals

Bortezomib (Velcade) and MLN2238 were provided by Millennium Pharmaceuticals, Inc

(Cambridge, MA, USA). Melphalan was purchased from Sigma-Aldrich (Madrid, Spain).

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)

were obtained from Peprotech (London, UK). All cell culture media and reagents were supplied

by Gibco (Paisley, UK). Primary antibodies were directed against: p65, ERK, β-catenin,

dephospho-β-catenin, phospho-GSK3β, GAPDH and IRE1α (Cell Signaling Technology;

Danvers, MA, USA), phospho-ERK (Santa Cruz Biotechnology; Santa Cruz, CA, USA), α-

tubulin (Calbiochem; Damstadt, Germany), T-cell factor 4 (TCF4; TCF7L2) (Millipore; Billerica,

MA, USA) and CD51/61 (R&D Systems; Minneapolis, MN, USA). Rhodamine-conjugated

phalloidin was purchased from Invitrogen (Carlsbad, CA, USA).

Cell cultures

Human MM cell lines MM.1S and MM.1R were provided by Dr Steven Rosen (Northwestern

University, Chicago, IL, USA). The MM.1S-luc cell line was a gift from Dr CS Mitsiades (Boston,

MA,USA), whereas RPMI8226 cells purchased from the American Type Culture Collection were

lentivirally transduced to stably express firefly luciferase as in Groen et al. (24). Cell viability of

MM cells in monoculture was assessed by MTT assay as previously described (19).

The human mesenchymal stem cell line immortalized by expression of the telomerase

reverse transcriptase gene (hMSC-TERT) was obtained from Dr D Campana (Memphis, TN,

USA). Primary MSCs derived from the BM of healthy donors (n=5) and MM patients with (n=5)

or without (n=3) osteolytic lesions were generated as described by Garayoa et al (25). These

studies were performed with the approval of the Hospital Universitario de Salamanca review

board, and samples were obtained after written informed consent of subjects.

In vitro OC formation, resorption pits, F-actin ring formation and CD51/61 expression

Peripheral blood mononuclear cells (PBMCs) from healthy donors were differentiated by culture

in osteoclastogenic medium (containing 25 ng/ml M-CSF and 50 ng/ml RANKL) as described in

Garcia-Gomez et al (26). Alternatively, PBMCs from myeloma patients were also used. Assays




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related to OC formation and function included: RANKL-induced NF-κB activation, F-actin ring

formation and CD51/61 (αVβ3) expression (pre-OCs, 14 days differentiation); resorption

capacity (17 days differentiation) and OC formation (21 days differentiation), and were

performed as in Hurchla et al (27).

In vitro OB differentiation, alkaline phosphatase (ALP) activity and mineralization assays

OBs were generated from mesenchymal osteoprogenitors by culture in osteogenic medium

(containing 5 mM β-glycerophosphate, 50 µg/ml ascorbic acid and 80 nM dexamethasone) and

assayed as in Garcia-Gomez et al (26). Briefly, primary MSCs (passage 2-3) or the hMSC-

TERT cell line were exposed to different MLN2238 or bortezomib concentrations and cultured in

osteogenic medium for 11 or 21 days for ALP activity and matrix mineralization assays,

respectively. ALP activity was quantified by hydrolysis of p-nitrophenylphosphate into p-

nitrophenol (Sigma-Aldrich), whereas mineralization was assessed by quantitative

measurement of Alizarin Red staining.

Real-time RT-PCR analyses

The hMSC-TERT cell line was cultured for 14 days in osteogenic medium (pre-OBs) and total

RNA was isolated using TRIzol reagent (Invitrogen). TaqMan Gene Expression Assays (Applied

Biosystems; Foster City, CA, USA) were performed according to manufacturer’s instructions:

Runx2, Hs01047976_m1; Osterix, Hs00541729_m1; Osteopontin, Hs00959010_m1;

Osteocalcin, Hs01587814_g1 and DKK-1, Hs00183740_m1. The endogenous expression of

GAPDH was used for normalization and relative quantification of target gene expression was

calculated by the comparative threshold cycle method.

TCF4 luciferase reporter assay

MSCs (3 x 105) were transiently co-transfected with either 400 ng/ml TOPflash or FOPflash

firefly luciferase reporters [Addgene plasmids #12456-7 (28)] and 40 ng/ml pRL-SV40 Renilla

luciferase reporter (Promega; Madison, WI, USA), using Nucleofector II as per manufacturer´s

instructions (Lonza; Basel, Switzerland). After overnight recovery, cells were treated with or

without PIs for 24 hours and luciferase activity was evaluated using a Dual-Luciferase reporter




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system (Promega). The firefly luciferase/Renilla luciferase ratio was calculated to normalize for

transfection efficiency.

Gene silencing

hMSC-TERT cells were transfected (three times/week for 3 weeks) using SAFEctin-STEM

(Deliverics; Edinburg, UK) as per supplier´s instructions, either with ON-TARGETplus

SMARTpool siRNA targeting human IRE1 or ON-TARGETplus non-targeting pool as negative

control (Dharmacon; Lafayette, CO, USA).

Immunoblotting

Generation of protein lysates and Western blotting were performed following standard

procedures (26). A luminol detection system with p-iodophenol enhancement for

chemiluminescence was used for protein detection.

MM.1S-luc co-cultures

The hMSC-TERT cell line was plated overnight in 96-well plates (1.25 X 104 cells/well) and

MM.1S-luc cells (2 X 104/well) were then added and co-cultured for 48 hours in medium

supplemented with 0.5% FBS. Similarly, MM.1S-luc cells were added to previously generated

pre-OCs and maintained in the same low-serum medium for 5 days. MM.1S-luc growth was

assessed by measurement of luciferase activity.

Mouse model of BM disseminated human MM

Animal experiments were conducted according to institutional guidelines for the use of

laboratory animals and after acquired permission from the local Ethical Committee for animal

experimentation. Bortezomib and melphalan were solubilized in 0.9% saline whereas MLN2238

was prepared in a 5% solution of 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich). RPMI8226-luc

cells (5 X 106) were injected intravenously (i.v.) into 6-week-old NOD-SCID-IL2Rγ-/- mice

(Charles River; L´Arbresle, France) and tumor development was monitored by non-invasive

bioluminescence imaging (BLI) with a Xenogen IVIS 50 system (Caliper Life Sciences;

Alameda, CA, USA). After 3 weeks, animals were randomized into 4 groups (n=6/group)




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receiving: vehicle (5% solution of 2-hydroxypropyl-β-cyclodextrin, 3 times/week by oral gavage),

melphalan (2.5 mg/kg, 3 times/week by oral gavage), bortezomib [0.8 mg/kg, 3 times/week by

intraperitoneal (i. p.) injection] and MLN2238 (7.5 mg/kg, 2 times/week by oral gavage). Serum

levels of human Igλ (secreted by RPMI8226-luc cells) were also used as a measure of tumor

burden and determined by ELISA (Bethyl Laboratories; Montgomery, TX, USA).

Micro-computed tomography (micro-CT) analysis

One femur of each animal was fixed in 10% formalin and scanned using a micro-CT system

(MicroCATII; Siemens, Knoxville, TN, USA) as described previously (26). Analysis of trabecular

microarchitecture in the distal femur was performed using BoneJ (29), a bone morphometry

image analysis ImageJ plugin.

Bone turnover markers

Carboxy-terminal telopeptide collagen crosslinks (CTX, indicating bone resorption) and N-

terminal propeptide of type I procollagen (P1NP, indicating bone formation) were measured in

mice sera using ELISA systems (Immunodiagnostic Systems; Scottsdale, AZ, USA).

Histological and immunohistochemical analyses

The other femur from each animal was fixed in 10% formalin, decalcified and paraffin

embedded. Bone histological evaluation on paraffin sections was performed after hematoxylin-

eosin or immunohistochemical staining for TCF4 as previously described (26).

Statistical analyses

Each in vitro assay was performed at least three times using MSCs or PBMCs from different

individuals, and duplicates (RT-PCR) or triplicates were measured for each of the tested

conditions. Quantitative data are expressed as mean ± SD or SEM, as specified. The non-

parametric Mann-Whitney U test was used for comparison of vehicle control and specific

treatments, and differences considered statistically significant for values of p< 0.05 (SPSS

Statistics 15.0, Chicago, IL, USA).




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Results

MLN2238 inhibits in vitro myeloma cell growth even in the presence of microenvironment

support

The anti-MM activity of MLN2238 and bortezomib on various myeloma cell lines

(dexamethasone-sensitive MM.1S, dexamethasone-resistant MM.1R and RPMI8226) was

evaluated after a 48 hour culture by MTT assay (Fig. 1A, B). In agreement with previous reports,

half maximal inhibitory concentration (IC50) for MLN2238 ranged from ≅ 10 to 17 nM (19),

whereas bortezomib presented a more potent cytotoxic effect (IC50 ranging from ≅ 1.7 nM to 3

nM) for the same myeloma cell lines (Fig. 1A, B).

MSCs and OCs within the BM microenvironment have been reported to support growth

and survival of myeloma cells and to protect MM cells from chemotherapy-induced apoptosis (4,

30). We therefore tested the ability of MLN2238 to decrease the growth of MM.1S-luc cells

when grown in co-culture with MSCs (Fig. 1C) or OCs (Fig. 1D). As expected, higher doses of

both bortezomib and MLN2238 were necessary to inhibit MM.1S-luc cell growth in the co-

culture as compared to monoculture conditions. Overall, although MLN2238 clearly overcame

the proliferative advantage conferred by microenvironmental cells (Fig. 1C, D), higher MLN2238

concentrations were required to attain similar efficacy as bortezomib on myeloma cells.

MLN2238 inhibits in vitro osteoclastogenesis and resorption

To compare the relative potency of MLN2238 versus bortezomib on OC formation,

osteoclastogenic cultures were exposed to a wide range of concentrations of each agent along

the differentiation process. As previously shown (13), bortezomib markedly inhibited the

formation of TRAP+ multinucleated cells (IC50 ≅ 1.2 nM) derived from PBMCs of healthy donors.

MLN2238 was able to suppress osteoclastogenesis with similar efficacy but when added at

higher concentrations (IC50 ≅ 10.9 nM) (Fig. 2A). A higher sensitivity to MLN2238 inhibitory

effect on OC formation was observed when using PBMCs from myeloma patients (IC50 ≅ 4.8

nM; data not shown). Noteworthy, as observed in representative micrographs of Fig. 2A, cell

densities in our cultures at 10 nM MLN2238 (a concentration similar to the IC50 for this agent on




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OC formation) were not significantly reduced, thus suggesting a selective effect of MLN2238 on

inhibition of OC formation rather than an effect on cell viability.

We next examined the effect of MLN2238 on osteoclastogenic cultures established on

calcium substrate-coated slides. A marked and dose-dependent reduction of the area of

resorbed pits was observed with MLN2238 treatment (Fig. 2B), similarly to that already reported

for bortezomib (13, 31). Specifically for MLN2238, doses required to inhibit OC resorption were

well below the ones required to inhibit OC formation, thus suggesting that this PI not only

inhibits osteoclastogenesis but also has a particular effect on OC resorption.

It is known that early OC differentiation mediated by RANKL-RANK stimulation is

primarily mediated through the adapter protein TRAF6, which then leads to activation of

mitogen-activated protein kinases (MAPK) (i.e. c-Jun N-terminal kinase, p38 and Erk), NF-κB,

and activator protein-1 (32). To gain some insight into the mechanistics mediating MLN2238

effect in inhibition of OC formation and function, we first tested its effect on MAPK pathway

activation. As shown in Fig. 2C, both MLN2238 and bortezomib inhibited RANKL-stimulated

phosphorylation of Erk.

Downstream signaling after RANKL stimulation in OC precursors activates NF-κB

transcription factor (32), which is not only a key regulator of OC formation but also of OC

function and survival. Both MLN2238 and bortezomib targeted NF-κB signaling on pre-OCs,

preventing RANKL-induced translocation of NF-κB into the nucleus (Fig. 2D). This effect is

thought to be due to impaired degradation of I-κB (the natural NF-κB inhibitor) by the

proteasome, which then remains bound to NF-κB retaining it in an inactive form in the

cytoplasm (13).

Relative to other mechanisms mediating the inhibition of OC resorption, we assessed

that pre-OCs differentiated under 2.5 nM bortezomib and 10 nM MLN2238 (concentrations

below their respective IC50 for OC formation to allow osteoclastogenesis) were associated with a

partial or complete disruption of the F-actin ring (Fig. 2E), as has already been described for

other PIs (33). Furthermore, and related to this latter effect, MLN2238 at the same

concentration was capable of reducing the expression of the αVβ3 integrin (CD51/61) on viable

7-AAD- OC precursors (Fig. 2F). The reduced levels of αVβ3 integrin and the disruption of the




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F-actin ring would impair maintenance of the sealing zone and prevent an effective bone

resorption (34).

MLN2238 promotes in vitro osteogenic differentiation and matrix mineralization of

osteoprogenitor cells from myeloma patients

To check whether MLN2238 was capable of promoting OB differentiation and function in vitro,

primary MSCs from myeloma patients (5/8 with bone lesions) were allowed to differentiate in

osteogenic medium along with different concentrations of the agent, and ALP activity was

determined as a surrogate marker of early osteoblastic activation. As observed in Fig. 3A,

MLN2238 dose-dependently increased ALP activity in early stage OBs to levels similar to that

attained by bortezomib. Similarly to its effect on ALP activity, MLN2238 also augmented matrix

mineralization and calcium deposition when present along the complete osteogenic

differentiation process (Fig. 3B). MLN2238 and bortezomib were capable of increasing ALP

activity and/or mineralization of MSCs from both patients with osteolytic lesions (see

representative micrographs in Fig. 3B) and without osteolytic lesions, although basal levels of

these parameters without treatment were generally higher in OBs derived from the latter MSCs.

Likewise, increased ALP activity and matrix mineralization were observed with mesenchymal

progenitors derived from healthy donors (data not shown).

The relative expression of several late bone formation markers (namely, the

downstream Osterix transcription factor, osteopontin and osteocalcin) were also determined

after PI-treatment of MSCs from myeloma patients at concentrations yielding maximal induction

of ALP activity and bone mineral formation (Fig. 3C). Both PIs significantly augmented the

expression of the mentioned OB markers. Since bortezomib has been found to suppress Dkk-1

expression in mouse calvarial cultures and BM stromal cells (14), we tested whether a similar

effect could be obtained in our experimental setting. MLN2238 was also capable of achieving a

modest yet significant reduction of Dkk-1 mRNA levels in pre-OBs differentiated from MSCs

from myeloma patients (Fig. 3C).

MLN2238 activates TCF4/β-catenin signaling and the UPR response in mesenchymal

precursors




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Numerous studies have demonstrated a pivotal regulatory role for the canonical Wnt signalling

pathway in commitment of mesenchymal progenitors to the OB lineage, in OB function and in

skeletal development (35, 36). Upon binding of canonical Wnt ligands to appropriate receptors

in osteoprogenitors, β-catenin is stabilized and translocated to the nucleus where it interacts

with a member of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor

family, to synergistically regulate the expression of OB differentiating genes (i. e. Runx2) (35).

Since TCF4 is the most abundant TCF family transcription factor expressed in OB cell lines and

primary hMSCs (37), we explored whether MLN2238 treatment was able to enhance TCF4

transcriptional activity using a TOPflash reporter system. When MSCs from myeloma patients

were PI-treated for 24 hours, a dose-dependent increase in luciferase activity of the reporter

plasmid was observed (Fig. 4A). Therefore, activation of TCF4 transcriptional activity seems to

mediate, at least in part, MLN2238 promotion of osteoblastogenesis and OB function.

Stabilization and nuclear translocation of β-catenin is also used as a metric of enhanced

canonical Wnt signaling, and thus we also tested whether MLN2238 could modulate β-catenin

levels. As seen in Fig. 4B, MLN2238 treatment of MSCs from myeloma patients slightly

upregulated levels of dephosphorylated β-catenin, which is the stabilized active form of β-

catenin able to interact with TCF/LEF transcription factors. These increased stabilized β-catenin

levels may be ascribed to a modest inhibition of glycogen synthase kinase 3 beta (GSK3β)

activity due to increased phosphorylation (Fig. 4B) (38). Taken together, our data show that

MLN2238 treatment leads to increased TCF4 transcriptional activity along with modest

augmented levels of active β-catenin, thus leading to activation of TCF4/β-catenin signaling on

osteoprogenitor cells.

OBs produce large amounts of extracellular matrix proteins and must activate the

unfolded protein response (UPR) to handle the accumulation of misfolded proteins. In fact, the

IRE1α-XBP1 pathway of the UPR has been recently shown to be essential for OB differentiation

by driving the transcription of Osterix (39). Increased levels of IRE1α protein were readily

observed after bortezomib and MLN2238 treatment of hMSC-TERT cells (Fig. 4C). Conversely,

silencing of IRE1α markedly abrogated PI-enhanced mineralization (Fig. 4D, E), thus

underscoring the essential role of the IRE1α component of the UPR in the promotion of OB

function by PIs.




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MLN2238 prevents tumor-associated bone loss besides reducing MM tumor burden

We next explored whether pro-osteogenic, anti-osteoclastogenic and anti-myeloma in

vitro effects of MLN2238 could be translated into a disseminated murine model of human

myeloma (27). RPMI8226-luc cells were intravenously injected to NSG mice and myeloma

engraftment (as monitored by bioluminescence) occurred after 4 weeks. At this point mice were

randomized to receive vehicle, melphalan, bortezomib or MLN2238 treatments as indicated

(see Methods and Fig. 5A). Melphalan was employed as a control drug with expected anti-

myeloma activity but not a bone effect (17). Compared to the vehicle control group, both orally

administered MLN2238 and i.p. delivered bortezomib very efficiently controlled tumor

progression, as measured by bioluminescence (Fig. 5A) or by serum hIgλ levels secreted by

RPMI8226-luc cells (Fig. 5B). Melphalan treatment, at doses used in our study, reduced

myeloma burden at only about 50% of levels observed with MLN2238 or bortezomib (Fig. 5A,

B).

Representative microCT images at the methaphyses of distal femurs showed evident

tumor-associated bone loss in vehicle and melphalan-treated mice, in contrast to trabecular

structures observed in both MLN2238 and bortezomib-treated animals (Fig. 6A). This was also

reflected by bone morphometric parameters which resulted in increased trabecular bone volume

and connectivity and reduced trabecular separation only in PI-treated animals, as compared to

vehicle control (Fig. 6B-D). In agreement with these data, histologic sections of MLN2238 and

bortezomib-treated mice presented higher number of trabeculae at the growth plate of the distal

femur (Fig. 6E), together with immunoreactive TCF4 OBs lining the trabeculae (Fig. 6F). Of

interest, TCF4 positive OBs in PI-treated mice presented a square or rectangular section

indicative of active bone matrix secretion, whereas flat OBs in vehicle and melphalan groups

reflected a resting secretory state (Fig. 6G). Finally, these findings correlated with levels of bone

turnover markers analyzed in serum after drug treatment. The bone resorption marker CTX was

only significantly diminished in PI-treated animals (Fig. 6H). P1NP, a bone formation marker,

augmented significantly in the PI-treated groups versus the control group (Fig. 6I), whereas

melphalan-treated animals showed no change.




15

Discussion

Osteolytic lesions are the most common complication of myeloma, being developed in

more than 80% of myeloma patients. These lesions often lead to skeletal-related events (SREs)

including focal lytic lesions, pathologic fractures, hypercalcemia, severe bone pain and vertebral

compression fractures (2, 40). Importantly, MBD not only negatively impacts the quality of life of

myeloma patients, but has been found to be associated with decreased overall survival (41).

This highlights the need for supportive bone-targeting treatments besides anti-myeloma

chemotherapies to reduce the risk of bone complications in MM. Moreover, since many of the

factors dysregulated in MBD are also important contributors to myeloma progression and

chemoresistance, it is expected that restoring bone homeostasis would indirectly and

additionally lead to tumor inhibition (42).

Bisphosphonates (BP) are anti-catabolic agents representing the current standard

supportive treatment for myeloma patients with MBD. Being pyrophosphate analogs, BPs

become adsorbed to mineralized matrix and cause apoptosis when OCs incorporate them by

endocytosis (43). Second-generation nitrogen-containing BPs now in use, such as pamidronate

and zoledronic acid, have dramatically reduced the incidence of SREs and significantly

improved the quality of life in MM patients (40). Nevertheless, adverse side effects may result

from BP therapy, including osteonecrosis of the jaw, impaired renal function and accumulation

of bone microfractures (2, 44); in addition, MBD may progress during BP treatment, presumably

because these compounds inhibit bone resorption but do not restore new bone formation (3).

Recent progress in the understanding of the molecular interplayers involved in MBD has

stimulated the development alternate bone therapeutic compounds either focusing on OC

inhibition, or importantly, on osteoblastogenesis promotion [reviewed in (2, 40, 42)]. It is likely

that these agents, now at different stages of clinical development, will contribute to a more

efficacious and improved management of MBD in the near future.

Within current agents used in anti-myeloma chemotherapy, bortezomib is unique

because of its concurrent anti-myeloma, anti-catabolic and bone anabolic activities, as

evidenced in multiple preclinical studies and in bortezomib-treated patients (10, 13, 15, 17). This

represents an unprecedented advantage for the treatment of both MM and MBD either as single




16

agent (12) or in certain combinations with other anti-myeloma drugs (11). Preclinical studies

with second-generation PIs carfilzomib and ONX0912, have shown that in addition to their anti-

myeloma properties (45, 46) and similar to bortezomib, they effectively target OB and OC

populations to shift the bone microenvironment from a catabolic to an anabolic state (27). Akin

to these PIs, herewith we show that MLN2238 also combines anti-myeloma activity with anti-

resorptive and bone-forming effects on bone.

As observed in our in vitro experiments, MLN2238 markedly inhibited OC formation

from human PBMC progenitors as well as OC resorption. Mechanistically, these effects on OC

function were at least partially mediated through inhibition of RANKL-induced NF-κB signaling,

F-actin ring disruption and diminished expression of the αVβ3 integrin, molecular sequelae also

shared by bortezomib and other PI treatments (13, 31, 33). Proteasome inhibition has also been

linked to induced in vitro differentiation of MSCs into OBs and enhanced OB function (14, 15,

37). We present here that, in a similar range of concentrations affecting OCs, MLN2238 was

also able to stimulate the in vitro osteogenic differentiation of MSCs and to promote OB

function; of interest, MLN2238 effects were observed on MSCs irrespective of being derived

from healthy donors or myeloma patients (including those from patients with osteolytic lesions).

As already reported for bortezomib and carfilzomib (37, 47), MLN2238 dose dependently

increased TCF4 transcriptional activity in reporter assays on osteoprogenitors. In relation with

TCF4 transactivation, we also found that MLN2238 treatment was linked to modest increased

levels of active dephosphorylated β-catenin (35), all of which would enhance TCF/β-catenin

signaling and promote OB function. Interestingly, we have also shown that MLN2238 treatment

markedly augments levels of the IRE1α component of the UPR, and as evidenced by siRNA

strategies, reduced levels of this protein impaired matrix mineralization even in the presence of

PIs. This is in agreement with recent reports highlighting the critical role of the UPR, and

specifically the IRE1α-XBP1 pathway in OB differentiation through promotion of Osterix

transcription (39). Although both myeloma cells and MSCs/OBs are protein secretory cells

dependent on the UPR to eliminate misfolded proteins and to cope with ER stress, PI treatment

exerts apoptosis on the former and promotes osteogenic differentiation on the latter. The reason

for these differential effects may reside on the lower threshold of MM cells for PI-induced




17

terminal UPR and ER-stress induced apoptosis, since these cells already highly express pro-

survival UPR components to function as secretory cells (48).

Our studies on OCs and OBs, as well as those on myeloma cell lines alone or in co-

culture with microenvironment cells, clearly showed that MLN2238, although less potent,

reached a similar efficacy as bortezomib. This difference in potency may reside on the shorter

dissociation half-life of MLN2238 with respect to bortezomib for proteasome inhibition (8), thus

higher concentrations of MLN2238 being needed to attain an equivalent effect. Nevertheless,

clinical pharmacokinetic studies from once-weekly orally administered MLN9708 estimate

plasma C max ~ 150 nM (49), well over the required in vitro doses for MLN2238´s effect on OBs

and OCs in our studies. It is therefore conceivable that therapeutically administered MLN9708

will achieve anti-resorptive and pro-anabolic bone effects (besides its anti-myeloma activity) in

myeloma patients.

In fact, as observed in our in vivo murine model, MLN2238 and bortezomib treatments

were very efficient in controlling the growth of RPMI8226-luc tumor cells in the BM, as assessed

by decreased bioluminescence imaging and diminished serum levels of human IgGλ. Increased

bone trabecular architecture in MLN2238 and bortezomib treated animals was evident on

microCT images at metaphyses of distal femurs as compared to vehicle control, correlating with

significantly augmented bone parameters of trabecular bone volume and connectivity and

reduced trabecular separation. This global bone anabolic effect of MLN2238 and bortezomib

was also associated with: i) increased trabeculae at the growth plate as observed on

histological sections, ii) immunostaining for TCF4 and active morphological appearance of OB

cells lining the trabeculae, and iii) reduced CTX (bone resorption marker) and increased P1NP

(bone formation marker) serum levels. All this evidence clearly supports, in addition to our in

vitro data, that these PIs have a double direct effect on bone, both promoting bone formation

and inhibiting bone resorption. To exclude the possibility that MLN2238´s effects on bone were

a secondary consequence of its anti-myeloma activity, we compared the in vivo effect of

MLN2238 to that of melphalan, an agent with accepted anti-myeloma but no direct effects on

bone (17). Although with our specific in vivo melphalan dosing and schedule this compound was

only ≈ 50% as effective as bortezomib or MLN2238 in controlling tumor burden, its anti-

myeloma activity was not linked to an indirect effect on bone, with no significant changes




18

observed with respect to vehicle control on bone turnover serum markers, trabecular

architecture, OB histological appearance or TCF4 immunostaining. These data reinforce our

previous interpretation of bone effects of MLN2238 majorly being due to direct targeting of OB

and OC populations in the BM, albeit indirect bone beneficial consequences of its anti-myeloma

activity cannot be excluded.

Taken together, we have shown that clinically relevant concentrations of the next-

generation PI MLN2238, promote OB differentiation and function (even in osteoprogenitor cells

from MM patients bearing osteolytic lesions) and inhibits OC formation and resorption in ex vivo

experiments. In a mouse model of disseminated human myeloma, oral administration of

MLN2238 was found to be at least equally effective as i. p. administered bortezomib in the

control of myeloma growth and in prevention of bone loss, sustained by both bone-forming and

anti-catabolic activities. Our studies therefore provide rationale for the clinical evaluation of

MLN9708 in the treatment of MBD and other bone pathologies. Looking at the future, it is likely

that the next years will show the real value of MLN9708 and other second generation PIs, such

as carfilzomib and oprozomib, as both anti-MM and bone-directed agents.

Acknowledgements

We acknowledge Dr Randall Moon (Howard Huges Medical Institute, Chevy Chase, MD, USA)

for making M50 Super 8x TOPflash and M51 Super 8x FOPflash plasmids available at Addgene

non-profit plasmid repository. We are also indebted to Montserrat Martín, Susana Hernández-

García, Laura San Segundo, Isabel Isidro, Teresa Prieto and Almudena Martín (Centro de

Investigación del Cancer and Hospital Universitario de Salamanca) for their help and excellent

technical work.

Author contributions

Conception and design: AG-G; MC; EMO; DC; JFSM; NR and MeG.

Contributed with BM samples/patient information/material/technical advice: EMO, JFB,

AJB and ACMM.




19

Development of methodology and performance of micro-CT and histology studies: CO-d-

S, TH-I, ACMM, RWJG, JM-U, SF and MiG.

Performed experiments: AG-G; DQ; MC; ZT and MeG.

Analysis and interpretation of data: AG-G; MC; EMO; ZT; DC; JSFM; NR and MeG.

Writing, review and/or revision of the manuscript: AG-G; MC; AJB; DC; JFSM, NR and

MeG.

Figure legends

Figure 1. MLN2238 is cytotoxic to MM cells and effectively suppresses the BM

microenvironment proliferative advantage to MM cells. A-B) MM.1S, MM.1R and RPMI8226

cell lines were cultured in the presence of indicated concentrations of bortezomib or MLN2238

for 48h and cell viability was assessed by MTT assay. C-D) MM.1S-luc cells were co-cultured

with the hMSC-TERT cell line for 2 days (C) or pre-OCs derived from healthy donors for 5 days

(D) in medium supplemented with 0.5% FBS. After the co-culture period, MM.1S-luc growth was

assessed by luciferase bioluminescence signal. In all graphs, data represent the mean ± SD of

three independent experiments.

Figure 2. MLN2238 inhibits in vitro OC formation and resorption. A) PBMCs from healthy

donors were cultured in osteoclastogenic medium under continuous PI treatment for 21 days,

and OCs were identified as multinucleated (≥3 nuclei) TRAP+ cells. Graphs represent the mean

± SD of three independent experiments. Representative micrographs of TRAP+ OCs are

shown. B) To test the effect of MLN2238 on OC resorption, PBMCs were cultured on calcium-

coated slices in the presence of osteoclastogenic medium and continuous PI treatment as

specified. After 17 days, OCs were removed and the area of resorption lacunae analyzed. Data

are expressed as mean ± SD. Representative micrographs of resorptive pits after MLN2238

treatment are reported. A and B: *p<0.05, versus vehicle control. Bar = 50 μm. C) PBMCs were

differentiated in osteoclastogenic medium for 5 days and then exposed to 2.5 nM bortezomib or

10 nM MLN2238 for 24 hours. Both PIs inhibit RANKL-induced Erk1/2 phosphorylation as




20

shown by Western blot. D) Pre-OCs (14 days in osteoclastogenic medium) were cultured

overnight in the absence of RANKL, treated for 3 hours with PIs and stimulated with RANKL for

30 min prior to immunostaining for the p65 subunit of NFκB. In absence of PIs (vehicle), RANKL

induces p65 translocation to the nucleus, whereas after PI-treatment p65 subunit is retained in

the cytoplasm. The p65 subunit is visualized in red and the nuclei by DAPI staining.

Representative micrographs of each condition are shown. Bar = 12.5 μm. E) Pre-OCs

differentiated under drug exposure were stained with rhodamine-conjugated phalloidin and

examined by fluorescence microscopy to visualize the F-actin ring. Representative micrographs

are shown. Bar = 50 μm. F) In the same setting as in (E), flow cytometry analysis revealed a

decreased expression of CD51/61 (αVβ3-integrin) in 7-AAD- cells. Data are expressed as

percentage of mean fluorescence intensity (% MFI) ± SD. *p<0.05, versus vehicle control.

Figure 3. MLN2238 increases ALP activity, stimulates matrix mineralization and

augments the expression of bone formation markers in in vitro differentiating OBs. A)

Primary MSCs from myeloma patients were cultured in osteogenic medium in the presence or

absence of PIs at indicated concentrations and ALP activity was measured at day 11. Results

are expressed as mean ± SEM. B) Matrix mineralization was assessed by Alizarin Red staining

and subsequent dye quantification in OBs differentiated from MSCs from MM patients at day 21.

Graphs illustrate mean values ± SD. Representative images derived from MSCs of a patient

with osteolytic lesions are shown. C) Total RNA was isolated from primary MSCs from MM

patients differentiated towards the osteogenic phenotype for 14 days in the presence or

absence of bortezomib or MLN2238. Real-time RT-PCR was performed to determine the

expression of osteoblastogenic markers (Osterix, osteopontin and osteocalcin), together with

the expression of the Wnt signaling inhibitor DKK-1. Expression levels for each gene were

normalized to GAPDH expression and referred to those in the absence of PIs. Data are

represented as the mean ± SD. All panels: *p<0.05, versus vehicle control.

Figure 4. MLN2238 promotes OB differentiation and function through activation of TCF/β-

catenin signaling and the Unfolded Protein Response (UPR). A) Primary MSCs from

myeloma patients were transiently transfected with the TOPflash (containing 7 TCF/LEF binding




21

sites) or FOPflash (with 6 mutant TCF/LEF binding sites) luciferase reporters. One day after

transfection, MSCs in osteogenic medium were treated for 24 hours with indicated

concentrations of bortezomib or MLN2238 and luciferase activity was measured in cell lysates.

Results represent the mean ± SD. Negative control (NC) refers to luciferase activity after

transfection with the FOPflash reporter at 10 nM bortezomib and 100 nM MLN2238. B) MSCs

from MM patients were differentiatied for 7 days in osteogenic medium and treated with 2.5 nM

bortezomib or 10 nM MLN2238 for 72 hours. Levels of active (dephosphorylated) β-catenin and

inhibited (phosphorylated) GSK3β were evaluated by Western blot analysis. C) PI-treatment

(bortezomib 25 nM and MLN2238 100 nM) of hMSC-TERT cells over a 24 hour period in

osteogenic medium augmented IRE1α levels as determined by Western blot. D) Transfection of

hMSC-TERT cells with IRE1α targeting siRNAs (100 nM, 48 hours) effectively diminished

IRE1α expression at the mRNA and protein level. Negative control (NC) refers to IRE1α

expression after transfection with non-targeting siRNAs. E) The hMSC-TERT cell line was

maintained in osteogenic medium for 21 days and transfected 3 times/week with IRE1α or NC

siRNAs. When IRE1α was silenced matrix mineralization was abolished even in the presence of

PIs (5 nM bortezomib; 10 nM MLN2238). Data represent the mean ± SD of at least three

independent transfections for each condition, each of them run in triplicate. For (A), (D) and (E):

p*<0.05 versus vehicle or NC.

Figure 5. Oral administration of MLN2238 decreased tumor burden in a mouse model of

disseminated MM. RPMI8226-luc cells (5 X 106) were i. v. injected into NSG mice. After 3

weeks, mice were randomized into 4 groups [receiving vehicle (MLN2238 vehicle), melphalan,

bortezomib and MLN2238; n = 6/group], and treated for additional 4 weeks with dosing and

regimen schedules as specified. Both i. p. administered bortezomib and orally delivered

MLN2238 effectively decreased tumor burden as measured by bioluminescence imaging (A)

and levels of serum human Igλ secreted by RPMI8226-luc cells (B). At doses and administration

regimens used in this study, melphalan was less effective than PIs in controlling tumor

progression. All data (A, B) are represented as mean ± SD. In (A): p**<0.01 bortezomib versus

vehicle control at each time point; p##<0.01 MLN2238 versus vehicle control at each time point;

in (B): p*<0.05 versus vehicle control group.




22

Figure 6. MLN2238 prevented myeloma-associated bone loss. A) Representative microCT

cross-sections at the metaphyses of distal femurs show loss of trabecular architecture in vehicle

and melphalan-treated mice, but not in mice treated with bortezomib or MLN2238. B-D) As

compared to vehicle control animals, MLN2238 significantly increased bone parameters of

trabecular bone volume and connectivity whereas trabecular bone separation was decreased.

Results are expressed as mean ± SD. p*<0.05 versus vehicle control. E-F) Representative

femur sections after drug treatment stained with hematoxylin and eosin (E), or for TCF4

immunohistochemistry (F). Bar = 25 µm. Dotted line delimits infiltrating MM.1S-luc cells and

host tissue. OB-like cells immunostained for TCF4 can be observed lining the trabeculae

(arrowheads). G) Representative images at higher magnification showing flat OBs lining the

trabeculae of vehicle-treated animals (arrows), whereas OBs were cuboidal in shape and

presented TCF4 immunostaining in MLN2238-treated femurs (arrowheads). Bar = 12.5 µm. H-I)

MLN2238 significantly changed bone turnover markers in serum, increasing levels of N-terminal

propeptide of type I procollagen (P1NP), a bone formation marker, while decreasing levels of

carboxy-terminal telopeptide collagen crosslinks (CTX), a bone resorption marker, as compared

with vehicle-treated animals. (H, I): Graphs illustrate mean values ± SEM. p*<0.05 p**<0.01

versus vehicle or between indicated groups.

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r

l a t i

e

v

t

o

c

n

o t r

l

o )

0 1 2.5 5 10 25 0 1 2.5 5 10 25 0

5 0

10 0

15 0

% L

c

a

n

u

e

a

e

r

a

a

( e

r

l a t i

e

v

t

o

c

n

o t r

l

o )

0 1 2.5 5 0 2.5 10 25 0

5 0

10 0

15 0

TRAP+ osteoclast formation Bone resorption

A B

*

*

* * *

*

*

* *

*

*

*

*

*

bortezomib (nM) MLN2238 (nM) bortezomib (nM) MLN2238 (nM)

MLN2238

vehicle 2.5 nM 10 nM 25 nM

MLN2238

vehicle 2.5 nM 10 nM 25 nM

vehicle bortez 2.5 nM C D

E

p6

5

MLN2238 10 nM

DA

PI

Me

rge

1

2.5

2.5

10

αVβ3 integrin (CD51/61)

Proteasome

inhibitors

vehicle

bortezomib

MLN2238

%MFI ± SD

100 0

nM

81.28 ± 21.77

64.51 ± 15.96

76.05 ± 18.62

70.10 ± 15.87

F

vehicle bortez 2.5 nM MLN2238 10 nM

MLN2238

bortez

vehicle

Day 5

p-Erk1/2 42/44 KDa

42/44 KDa Erk1/2

GAPDH 37 KDa

*

*Research. on December 18, 2020. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from



A L

P

c

a

t i v i t

y (

n M

/ m n

i

/ μp

g

r o t e

n

i )

Figure 3, Garcia-Gomez et al.

A i

l z a r

n

i

R e

d ( m

M )

Mineralization

0 1 2.5 5 0 2.5 5 10


A B

C

O sterix Osteocalcin Osteopontin DKK-1 0

1

2

3

n

o

i s

s

e

r

p

x

e

e

v

i t

a

l e

r

s

r e

k

r

a

m

e

n

o

B

v ehi c l e

b o r t e z o m ib 5 nM

M L N 2238 10 nM

*

* * *

*

* *

*

*

* * *

*

*

*

* *

*

* *

OB markers

10 nM

5 nM

vehicle

MLN2238

0

10

20

30

40

50

ALP activity

0.0

0.5

1.0

1.5

2.0

0 1 2.5 5 0 2.5 5 10





TCF4 activity

bortez MLN2238

6 12 24 6 12 24

veh

IRE1α

α-tubulin 60 KDa

110 KDa

C

A


D

0

500

1000

1500

2000

2500R

ela

tiv

e L

uc

ife

ras

e U

nit

s

0 5 10 25 NC 0 50100250NC

*

* *

*

*

*

0

50

100

siRNA NC IRE1

NC IRE1

IRE1α 110 KDa

α-tubulin 60 KDa

siRNA

IR

E1

α

re

lati

ve

ex

pre

ss

ion

vehicle bortez MLN2238

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

NC IRE1 NC IRE1 NC IRE1

Aliza

rin

Re

d (

mM

)

siRNA

E

MLN2238

bortez

vehicle

deP-βcatenin 92 KDa

92 KDa βcatenin

p-GSK3β 46 KDa

37 KDa GAPDH

B


*

** *




0 4 7 10 14 18 22 25 28 0.0

0.5

1.0

1.5

2.0

2.5

vehicle

melphalan 2.5 mg/kg p.o. (day 1,3,5) 4w

bortezomib 0.8 mg/kg i.p. (day 1,3,5) 4w

MLN2238 7.5 mg/kg p.o. (day 1,4) 4w

vehicle bortez MLN2238

1.0 2.0 3.0 4.0

Radiance

(photons/sec/cm2/sr)

x 10 6

hIg

Gλ

(n

g/m

l)

melph

T

ota

l fl

ux

(x1

0 p

ho

ton

s/s

ec

)

1

2


Days of treatment

0

500

1000

1500

Serum human IgGλ

MLN

2238

bortez

vehi

cle

* *

*

mel

ph

BLI imaging

A B

** ** **********## ## ## ## ## ## ##





0

5

10

15

20

B

MicroCT imagingMicroCT parameters

H Serum CTX

(bone resorption)

I Serum P1NP

(bone formation)

CT

X (

ng

/ml)

*

*

**

MLN

2238

bortez

vehi

cle

mel

ph

0

50

100

150

200

P1

NP

(n

g/m

l)

MLN

2238

bortez

vehi

cle

mel

ph

*

*

MLN

2238

bortez

vehi

cle

mel

ph

BV

/TV

0.0

0.2

0.4

0.6

Trabecular Bone

Volume

*

*

0.0

0.1

0.2

0.3

0.4

0.5

MLN

2238

bortez

vehi

cle

mel

ph

Trabecular

Separationvehicle

melph

bortez

MLN2238

A

E

He

ma

tox

yli

n &

eo

sin

s

tain

ing

T

CF

4

sta

inin

g

F

*

*

Connectivity

bortezomib MLN2238melphalan

Tb

.Sp

(m

m)

0

50

100

150

200

250

MLN

2238

bortez

vehi

cle

mel

ph

*

*

C D

Co

nn

ec

tiv

ity

bortezomibvehicle MLN2238melphalan

vehicle

G

MLN2238

vehicle

TCF4 staining




Published OnlineFirst January 31, 2014.Clin Cancer Res Antonio Garcia-Gomez, Dalia Quwaider, Miriam Canavese, et al. myeloma bone diseasePreclinical activity of the oral proteasome inhibitor MLN9708 in

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