HHS Public Access Marizomib and the IMiD Br J …triphaseco.com/wp-content/uploads/2017/04/Das_2015.pdfSynergistic Anti-Myeloma Activity of the Proteasome Inhibitor Marizomib and the

Synergistic Anti-Myeloma Activity of the Proteasome Inhibitor Marizomib and the IMiD® Immunomodulatory Drug Pomalidomide

Deepika Sharma Das1, Arghya Ray1, Yan Song1, Paul Richardson1, Mohit Trikha2, Dharminder Chauhan1,¶,*, and Kenneth C. Anderson1,¶,*

1LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

2Triphase Accelerator, San Diego, CA

Abstract

The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and

refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with

toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel

proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms

of action and effects on proteasomal activities, and that it can overcome bortezomib resistance.

Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved

by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that

combining low concentrations of marizomib with pomalidomide induces synergistic anti-MM

activity. Marizomib plus pomalidomide-induced apoptosis is associated with: 1) activation of

caspase-8, caspase-9, caspase-3 and PARP cleavage; 2) downregulation of cereblon (CRBN),

IRF4, MYC and MCL1; and 3) suppression of chymotrypsin-like, caspase-like, and trypsin-like

proteasome activities. CRBN-siRNA attenuates marizomib plus pomalidomide-induced MM cells

death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and

tumour-associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow

microenvironment. In human MM xenograft model studies, the combination of marizomib and

pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical

studies provide the rationale for on-going clinical trials of combined marizomib and

pomalidomide to improve outcome in patients with RRMM.

¶Address correspondence: Kenneth C. Anderson and Dharminder Chauhan, Dana-Farber Cancer Institute, Dept. of Medicine, Harvard Medical School, 44 Binney Street, Mayer 557, Boston, MA 02115, USA, Tel: +1 617-632-2144, Fax: +1 617-632-6140, [email protected]; [email protected].*Joint Senior authorship

Authors’ contributions: DSD performed the experiments, interpreted data and wrote the manuscript; YS and AR helped in animal studies; PR provided clinical samples; MT reviewed the manuscript; DC designed research, analysed data and wrote the manuscript; KCA analysed data and wrote the manuscript.

Conflict-of-interest disclosure MT is an employee of Triphase Accelerator; KCA is on Advisory board of Celgene, Onyx, Gilead, Sonofi Aventis and is a Scientific founder of Acetylon and Oncopep; DC is consultant to Triphase Accelerator. The remaining authors declare no competing financial interests.

Supplementary information is available online.

HHS Public AccessAuthor manuscriptBr J Haematol. Author manuscript; available in PMC 2016 December 01.

Published in final edited form as:Br J Haematol. 2015 December ; 171(5): 798–812. doi:10.1111/bjh.13780.

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Keywords

Marizomib; Pomalidomide; Multiple myeloma; cancer and drug therapy

Introduction

The ubiquitin proteasome pathway (UPP) is a validated therapeutic target in multiple

myeloma (MM), evidenced by the US Food and Drug Administration (FDA) approval of

bortezomib (Velcade™) and carfilzomib (Kyprolis™).(Kane, et al 2003, Richardson, et al

2003, Richardson, et al 2005, Siegel, et al 2012, Vij, et al 2012a, Vij, et al 2012b) Even

though bortezomib and carfilzomib therapies are major advances, they are associated with

possible off-target toxicities and the development of drug-resistance.(Atrash, et al 2015, Cai,

et al 2014, Harvey 2014, Huber, et al 2015, Lonial, et al 2005, Richardson, et al 2006,

Wanchoo, et al 2014) Our previous studies showed that the novel proteasome inhibitor

marizomib(Feling, et al 2003) is distinct from bortezomib and triggers apoptosis even in

MM cells resistant to bortezomib therapies.(Chauhan, et al 2005a) These preclinical data

provided the basis for the on-going phase-1 clinical trials of marizomib in patients with

relapsed/refractory MM (RRMM).(Potts, et al 2011, Richardson, et al 2011) In addition, we

showed that the combination of marizomib with the immunomodulatory agent lenalidomide

induces synergistic anti-MM activity.(Chauhan, et al 2010)

Pomalidomide, like lenalidomide, is a thalidomide analogue with potent immunomodulatory

activity. Based on increased progression-free survival(Gras 2013, Richardson, et al 2013,

Richardson, et al 2014), pomalidomide has been approved by the FDA for the treatment of

patients with RRMM who have received at least two prior therapies, including lenalidomide

and bortezomib, and who showed disease progression on or within 60 days of completion of

the most recent therapy.(Gras 2013, Richardson, et al 2013, Richardson, et al 2014)

In the present study, we characterize the effects of the combination of marizomib and

pomalidomide treatment against MM cell lines and primary patient cells resistant to

conventional and novel therapies. Both in vitro models and in vivo MM xenograft models

demonstrate that marizomib plus pomalidomide trigger synergistic anti-MM activity and

overcome drug resistance. Our preclinical studies support the continuation of clinical trials

of combined marizomib and pomalidomide to improve outcome in patients with RRMM.

Materials and methods

Cell culture and reagents

Human MM cell lines MM.1S, MM.1R, INA-6, ARP-1, RPMI-8226, DOX40, LR5,

ANBL-6.WT (wild type), and ANBL-6-bortezomib-resistant (ANBL-6.BR), as well as

peripheral blood mononuclear cells (PBMCs) from normal healthy donors, were cultured in

RPMI-1640 medium supplemented with complete medium (10% fetal bovine serum, 100

units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine) at 37°C and 5% CO2.

ANBL-6.WT and ANBL-6.BR cell lines were kindly provided by Dr. Robert Orlowski (MD

Anderson Cancer Center, Texas). Bone marrow stromal cells (BMSCs) were cultured in

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Dulbecco’s modified Eagle medium supplemented with complete medium. Patient CD138+

MM cells, BMSCs and plasmacytoid dendritic cells (pDC) were isolated and cultured as

described previously.(Chauhan, et al 2009) Informed consent was obtained from all patients,

in accordance with an Institutional Review Board approved clinical protocol. Marizomib

was obtained from Triphase Accelerator Corporation (San Diego, CA, USA), and

pomalidomide was purchased from Selleck chemicals (Houston, TX, USA).

Cytotoxicity and apoptosis assays

Cell viability in MM cell lines, patient MM cells and normal PBMCs were assessed by 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)/water-soluble

tetrazolium salt 1 (WST-1) assay. MM cell growth assessment in co-culture studies with

BMSCs or pDCs were performed using bromodeoxyuridine (BrdU) cell proliferation kits as

previously described.(Chauhan, et al 2008a) Apoptosis was quantified using FACSCanto

(BD Biosciences, San Jose, CA, USA). Caspase-8 and -9 fluorometric assay kits

(ALX-850-222-K101 and ALX-850-224-K101, Enzo Life Sciences, Farmingdale, NY) were

utilized to measure caspase-8 and caspase-9 enzymatic activity.

In vitro migration and capillary-like tube structure formation assays

The migration assay was performed using 24-well Transwell plates (Millipore, Billerica,

MA, USA) in the presence of 10% fetal bovine serum, and migrating cells were quantified

by measuring the fluorescence intensity, as previously described.(Chauhan, et al 2010)

Angiogenesis was determined in vitro by matrigel capillary-like tube structure formation

assay, as previously described.(Chauhan, et al 2008a) Human vascular endothelial cells

(HUVECs)(American Type Culture Collection [ATCC], Manassas, VA, USA) were

maintained in endothelial cell growth medium-2 supplemented with 5% FBS. After 3

passages, HUVEC viability was measured using Trypan blue exclusion assay; less than 5%

of cell death was observed with single or combined agents.

Western blotting and proteasome activity assays

Immunoblot analysis was performed using antibodies (Abs) against poly ADP ribose

polymerase (PARP) (BD Bioscience Pharmingen, San Diego, CA), cereblon (CRBN)

(Novus Biologicals), caspase-8, caspase-9, caspase-3, IRF4, MYC (c-Myc), MCL1,

HSP90AA1 (Hsp-90), HSPA1A (Hsp-70), HSPB1 (Hsp-27) or GAPDH (Cell Signaling,

Beverly, MA). Blots were then developed by enhanced chemiluminescence (Amersham,

Arlington Heights, IL). In vitro proteasome activity was performed using fluorogenic

peptide substrates, as previously described.(Chauhan, et al 2008a)

Transfection assays

CRBN knockdown experiment was performed using CRBN-siRNA (Origene, Rockville,

MD, USA). MM.1S cells were transfected with CRBN-siRNA or scrambled (scr)-siRNA

using the cell line Nucleofactor Kit V solution (Amaxa Biosystems/Lonza, Allendale, NJ,

USA), as per the manufacturer’s instructions. After cells were pre-treated with

pomalidomide for 24 h, marizomib was added for an additional 24h, followed by analysis of

cell viability by WST-1 assay.

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Human plasmacytoma xenograft model

The Animal Care and Use Committee of Dana Farber Cancer Institute approved all

experiments involving animals. The subcutaneous MM.1S mouse xenograft model was

generated as previously described(Chauhan, et al 2005a, Chauhan, et al 2008a, Chauhan, et

al 2010) CB-17 SCID-mice (n=30; Taconic, Petersburgh, NY, USA) were subcutaneously

inoculated with 5.0 × 106 MM.1S cells in 100 μl of serum-free RPMI 1640 medium.

Tumour size was measured in two dimensions using calipers; tumour volume was calculated

using the formula: V = 0.5 a x b2, where a and b are the long and short diameter of the

tumour, respectively. Animals were sacrificed when their tumours reached 2 cm3.

When tumours were measurable (~150 mm3), approximately 3 weeks after MM-cell

injection, mice (5 mice/group) were divided into random groups blindly and treated orally

with vehicle alone, marizomib (0.15 mg/kg), pomalidomide (0.5 mg/kg or 2.5 mg/kg) or

marizomib (0.15 mg/kg) plus pomalidomide (0.5 mg/kg or 2.5 mg/kg) for 24 days on a

twice-weekly schedule for marizomib and on 4 consecutive days weekly for pomalidomide.

Immunostaining

Sections of tumours harvested from mice were stained with anti-Ki67, anti-Caspase-3,

Factor VIII, or vascular endothelial growth factor receptor 1 (VEGFR1) Abs, as previously

described.(Chauhan, et al 2010) Immunostained tissues were imaged with a Zeiss

AxioImager M1 Microscope (Zeiss, Oberkochen, Germany) (Chauhan, et al 2010).

Statistical analysis

Statistical significance of differences observed in drug-treated vs. control culture was

determined using the Student’s t test. Statistical significance of differences observed in

tumour volumes in marizomib-, pomalidomide- or marizomib plus pomalidomide-treated

mice was determined using a Student’s t test. P values <0.05 were considered statistically

significant. Survival of mice was determined by GraphPad Prism software (GraphPad

Software, La Jolla, CA). Isobologram analysis(Chou and Talalay 1984) was performed by

using the CalcuSyn software program (Biosoft, Ferguson, MO, and Cambridge, United

Kingdom). A combination index value < 1.0 indicates synergism, and values > 1.0 indicate

antagonism.

Results

Anti-MM activity of combined low concentrations of marizomib and pomalidomide in vitro

Human MM-cell lines (MM.1S, INA-6, RPMI-8226, MM.1R, Dox-40, LR5, ANBL6.WT

and ANBL6.BR) were pretreated with pomalidomide for 24h; marizomib was then added for

an additional 24h, followed by assessment for cell viability using MTT assays. For the

combination studies, we utilized marizomib and pomalidomide across a range of

concentrations. An analysis of synergistic anti-MM activity using the method of Chou and

Talalay (1984) demonstrated a significant decrease in viability of all cell lines in response to

treatment with combined low concentrations of marizomib and pomalidomide compared

with either agent alone (Fig 1A–1D, and Supplementary Fig 1A–1E). Isobologram

analysis(Chou and Talalay 1984) confirmed synergistic anti-MM activity, with a

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combination index of < 1.0 in all MM cell lines tested. Importantly, the combination of low

concentrations of marizomib and pomalidomide overcame resistance to both conventional

and novel anti-MM agents. For example, marizomib plus pomalidomide decreased viability

of cell lines resistant to bortezomib (ANBL6.BR), dexamethasone (MM.1R), doxorubicin

(Dox-40) and melphalan (LR5) (Fig 1, and Supplementary Fig 1). Additionally, synergistic

anti-MM activity of combined marizomib and pomalidomide was also observed against

TP53-null ARP-1 MM cells (Fig 1C).

To determine whether the combination of low concentrations of marizomib and

pomalidomide similarly affected purified patient MM cells, we examined purified (CD138+)

MM cells from 2 newly diagnosed (Patients 6 and 7) and 5 patients with MM refractory to

multiple therapies (Fig 1E), including dexamethasone (Patient 1), bortezomib/

dexamethasone (Patients 2, 4 and 5), and lenalidomide/bortezomib/dexamethasone (Patient

3). MM was considered refractory to a specific therapy when disease progressed on that

therapy or relapsed within two months of discontinuing therapy. At the concentrations used,

either agent was moderately active in cells isolated from Patients 1 and 2 and was weakly

active or inactive in cells isolated from Patients 3–7. Importantly, a significant decrease in

cell viability of all patient MM cells was noted after combination therapy as compared to

either compound alone (p < 0.05 for all patients; Fig 1E). In contrast, marizomib (1.25 nM)

plus pomalidomide (2.5 μM) did not significantly decrease the viability of normal PBMCs

(Fig 1F), suggesting a favourable therapeutic index for this combination regimen.

Combined low concentrations of marizomib and pomalidomide block migration of MM cells and angiogenesis

Migration of MM cells and associated angiogenesis contributes to the progression of MM.

(Giuliani, et al 2003, Podar, et al 2001, Vacca, et al 2003) To determine whether marizomib

plus pomalidomide affects these processes, we utilized Transwell insert systems and in vitro

tubule formation assays. Serum alone increased MM.1S cell migration; importantly,

marizomib (1.25 nM) plus pomalidomide (2.5 μM) inhibited serum-dependent MM.1S cell

migration, as evidenced by a marked decrease in the number of migrating crystal violet-

stained cells (Fig 2A). Cells were >90% viable before and after performing the migration

assay, excluding the possibility that inhibition of migration was due to cell death. Our

findings show that marizomib plus pomalidomide blocked MM cell migration, which may

prevent homing of MM cells to the BM.

To determine whether marizomib plus pomalidomide exhibits anti-angiogenic activity, we

utilized in vitro capillary-like tube structure formation assays. Specifically, we performed

matrigel capillary-like tube structure formation assays: HUVECs plated onto matrigel

differentiate and form capillary-like tube structures reflecting in vivo neovascularization, a

process dependent on cell-matrix interaction, cellular communication and cellular motility.

Treatment of HUVECs with combined low concentrations of marizomib and pomalidomide,

but not either agent alone, significantly decreased tubule formation (Fig 2B). HUVEC

viability was assessed using the trypan blue exclusion assay and < 10% cell death was

observed with either single agents or the combined treatment. These findings suggest that

the combination of marizomib and pomalidomide block angiogenesis.

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Combination of low concentrations of marizomib plus pomalidomide inhibits bone marrow stromal cells (BMSCs)- and plasmacytoid dendritic cells (pDCs)-induced growth of MM cells

The MM-host BM microenvironment confers growth, survival and drug resistance in MM

cells.(Anderson 2007, Chauhan, et al 1996) Therefore, we next examined whether the

combination of marizomib plus pomalidomide retains its anti-MM activity in the presence of

the MM BM milieu. MM.1S cells were cultured with or without patient BMSCs in the

presence or absence of marizomib, pomalidomide, or marizomib plus pomalidomide. Low

concentrations of marizomib plus pomalidomide significantly inhibited BMSC-induced

growth of MM.1S cells, as assessed by WST-1 assays (Fig 2C).

Our recent studies have identified an integral role of pDCs in MM pathogenesis.(Chauhan,

et al 2009, Ray, et al 2015, Ray, et al 2014) The functional significance of increased

numbers of pDCs in the MM BM is evidenced by our observations that pDCs are relatively

resistant to novel and conventional therapies, protect tumour cells from therapy-induced

cytotoxicity, promote tumour growth and survival as well as suppress immune responses.

(Chauhan, et al 2009, Ray, et al 2015, Ray, et al 2014) We therefore examined the effect of

marizomib plus pomalidomide on pDC-induced MM cell growth. MM.1S cells (5 x 104

cells/200 μl) and patient pDCs (1 x 104 cells/200 μl) were cultured either alone or together.

Cells were pre-treated with pomalidomide for 24 h; marizomib was then added for an

additional 24 h; and growth was measured using a WST-1 assay. A significant inhibition of

pDC-induced MM.1S cell growth was observed in response to treatment with marizomib

plus pomalidomide (Fig 2D). Together, our data suggests that the combination of marizomib

and pomalidomide has the ability to overcome BMSC- or pDC-mediated MM cell growth

and drug resistance.

Mechanisms mediating the anti-MM activity of marizomib plus pomalidomide

As shown in Fig 1, the combination of low concentrations of marizomib plus pomalidomide

decreased the viability of MM cell lines and patient cells. This decrease in viability was due

to induction of apoptosis, as determined by Annexin V/propidium iodide staining and flow

cytometry (Supplementary Fig 2). Moreover, marizomib plus pomalidomide-induced

apoptosis in MM cell lines (MM.1S, RPMI-8226, Dox-40) was associated with PARP

cleavage (Fig 3A) and the activation of caspase-3, caspase-8 and caspase-9 (Fig 3B).

Biochemical inhibitors of caspase-3, caspase-8, caspase-9 or pan-caspases abrogated

marizomib plus pomaldiomide-induced cytotoxicity (Fig 3C). Of note, blockade of either

caspase-8 or caspase-9 led to a similar degree of inhibition of marizomib plus

pomalidomide-induced apoptosis.

Effects of marizomib plus pomalidomide on chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteasome activities

The primary target of proteasome inhibitors is the proteasome, which mediates degradation

of proteins via three (CT-L, T-L and C-L) proteolytically active sites.(Arendt and

Hochstrasser 1997, Heinemeyer, et al 1997) Therefore, we next examined the effect of

combined marizomib and pomalidomide on CT-L, C-L, and T-L activity. The combination

of low concentrations of marizomib (1.25 nM) and pomalidomide (2.5 μM) induced a

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marked inhibition of all three proteasomal activities versus either agent alone at these

concentrations (Fig 4A), suggesting that pomalidomide enhances the ability of marizomib to

target all three proteasome activities.

Effect of marizomib plus pomalidomide on cereblon and associated downstream signaling

The primary target of pomalidomide is the E3 ubiquitin ligase, cereblon (CRBN).(Lu, et al

2014) Immunoblot analysis of CRBN expression in MM cells and normal healthy donor

PBMCs showed significantly higher CRBN expression in MM cell lines than in normal

PBMCs (Fig 4B). We therefore examined the functional role of CRBN during marizomib

plus pomalidomide-induced cytotoxicity using loss-of-function studies. The functional

specificity of CRBN-siRNA was confirmed by a marked decrease in CRBN levels (Fig 4C,

immunoblot). Importantly, transfection of CRBN-siRNA, but not negative-control

(scrambled) siRNA significantly inhibited marizomib plus pomalidomide-induced apoptosis

in MM.1S cells (Fig 4C; p < 0.005). We found no statistically significant difference upon

treatment with marizomib alone in either CRBN siRNA or scrambled siRNA transfected

cells. Non-transfected cells showed results similar to those noted in scrambled siRNA

transfected cells (data not shown). These findings suggest that marizomib plus

pomalidomide-triggered apoptosis in MM cells is mediated, at least in part, via CRBN.

CRBN is linked to the activation of the downstream IRF4/MYC signalling axis.(Thakurta, et

al 2014) IRF4 is a survival factor for MM cells, and MYC is a direct target of IRF4 in

activated B cells and MM cells. Conversely, IRF4 is also a direct target of MYC

transactivation, generating an autoregulatory circuit in MM cells.(Shaffer, et al 2008)

Importantly, MYC coordinates cellular growth, metabolism and proliferation.(Holien, et al

2012) In this study, treatment of MM.1S and RPMI-8226 cells with marizomib plus

pomalidomide led to reduction of CRBN, IRF4 and MYC protein expression (Fig 4D and

Supplementary Fig 3). NF-κB (NFKB1) is another key mediator of MM cell survival.

(Chauhan, et al 1996) Interestingly, lenalidomide inhibits IRF4, which in turn

downregulates NF-κB in a CRBN-dependent manner.(Zhang, et al 2013) In our study,

treatment of MM cells with low doses of marizomib and pomalidomide also blocks NF-κB

activity (Supplementary Fig 4). Together, these findings suggest that marizomib plus

pomalidomide-induced apoptosis in MM cells is associated with downregulation of the

CRBN/IRF4/MYC signalling axis.

We next examined whether the combination of marizomib and pomalidomide overcomes

mechanism(s) that confer survival and drug-resistance in MM cells. For example, the anti-

apoptotic protein MCL1 confers drug resistance in MM cells(Fan, et al 2014), and we found

a marked decrease in MCL1 levels in marizomib and pomalidomide-treated cells versus

cells treated with either agent alone (Fig 4D). In addition, prior studies link heat shock

proteins (HSPB1, HSPA1A, HSP90AA1) with drug resistance.(Chauhan, et al 2005b,

Chauhan, et al 2003, Fribley, et al 2004, Landowski, et al 2005, Obeng, et al 2006)

Importantly, we found that the combination of low concentrations of marizomib and

pomalidomide did not induce HSPB1, HSPA1A or HSP90AA1 protein levels (Fig 4E).

Given the role of MCL1 and heat shock proteins in the development of drug-resistance, our

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data suggest that combined low doses of marizomib and pomalidomide may result in less

frequent occurrence of drug resistance in MM.

Combined marizomib and pomalidomide inhibit human MM cell growth in vivo and prolongs survival in the MM.1S MM xenograft mouse model

Having defined the efficacy of combined marizomib and pomalidomide in targeting MM

cells in vitro, we next validated these findings in vivo using our murine xenograft model of

human MM.(Chauhan, et al 2008a, Chauhan, et al 2010) This model has been useful in

validating the novel anti-MM therapies bortezomib and lenalidomide, leading to their

translation to clinical trials and FDA approval for the treatment of MM. A marked reduction

in tumour growth, as well as an increased prolongation of survival, was observed in

marizomib plus pomalidomide-treated mice versus mice receiving either agent alone (Fig

5A and 5B). These data demonstrate the effectiveness of the anti-tumour activity of

marizomib plus pomalidomide in vivo. The combination of marizomib and pomalidomide

treatment was well tolerated, as evidenced by the lack of weight loss even after 3 weeks of

treatment (Fig 5C). Blood chemistry profiles of marizomib plus pomalidomide-treated mice

showed that haemoglobin, bilirubin and creatinine levels were not statistically significant

altered (Fig 5D). Together, these findings suggest that combining marizomib and

pomalidomide markedly reduced tumour growth and is well tolerated in vivo.

We next examined the apoptotic activity of this drug combination in vivo using

immunostaining for caspase-3 activation and terminal deoxynucleotidyl transferase dUTP

nick end labeling (TUNEL) staining of tumours harvested from mice treated with vehicle

alone, marizomib, pomalidomide, or marizomib plus pomalidomide. The combination of

marizomib plus pomalidomide increased the number of cleaved caspase-3- and TUNEL-

positive apoptotic tumour cells compared to treatment with vehicle or either agent alone (Fig

6A and 6B). Furthermore, a decrease in the proliferation marker Ki67 was also noted in

tumours excised from mice receiving marizomib plus pomalidomide (Fig 6C). In concert

with our in vitro data (Fig 4D), tumours from mice treated with marizomib plus

pomalidomide showed decreased MCL1 and IRF4 levels (Fig 6D).

MM cell growth is associated with angiogenesis, and vascular endothelial growth factor

(VEGF) plays a role in this process.(Anderson 2012) Our in vitro data showed that the

combination of marizomib and pomalidomide exerts anti-angiogenic activity (Fig 2A and

2B). To determine whether marizomib and pomalidomide trigger anti-angiogenic activity in

vivo, we evaluated tumours harvested from mice by immunostaining using two distinct

markers of angiogenesis, Factor VIII and VEGFR1. As seen in Fig 6E, low doses of

marizomib or pomalidomide alone triggered a very modest decrease in the number of Factor

VIII-positive cells compared to sections from control-treated tumours, whereas combination

treatment decreased the number of Factor VIII-positive cells. Similarly, marizomib plus

pomalidomide markedly decreased the number of VEGFR1-positive cells (Fig 6F). These

data suggest that marizomib plus pomalidomide inhibits tumour-associated angiogenic

activity.

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Discussion

We show that the combination of low concentrations of marizomib and pomalidomide

decreased viability of MM cell lines and primary patient tumour cells, without affecting

normal PBMC viability. Our data show the anti-MM activity of marizomib plus

pomalidomide against MM cell lines, including those sensitive and resistant to conventional

and novel therapies, as well as representing distinct cytogenetic profiles.(Bergsagel, et al

1996, Bergsagel and Kuehl 2005) For example, we studied isogenic cell lines

dexamethasone-sensitive MM.1S(Greenstein, et al 2003) and dexamethasone-resistant MM.

1R with t(14;16) translocation and MAF (c-maf) overexpression; INA-6, an interleukin 6–

dependent cell line with NRAS activating mutation; and RPMI-8266 with TP53, KRAS and

EGFR mutations.(Avet-Loiseau, et al 2007, Bergsagel, et al 1996, Bergsagel and Kuehl

2005, Burger, et al 2001, Davies, et al 2003, Greenstein, et al 2003) Although combined

treatment with marizomib and pomalidomide decreased viability in all MM cell lines, the

50% inhibitory concentration of this combination regimen was different for each cell line,

which may be due to their distinct genetic heterogeneity and/or drug-resistance

characteristics.

We observed maximal synergistic anti-MM activity of combined marizomib and

pomalidomide when MM cells were pretreated with pomalidomide for 24 h followed by

addition of marizomib for another 24 h versus the simultaneous addition of these agents

(data not shown). It is likely that pomalidomide induces distinct apoptotic signalling versus

marizomib and/or upregulates proteasome load in MM cells, which together primes the cells

to undergo robust cell death in response to subsequent treatment with even low

concentrations of marizomib.

To determine whether the combination of marizomib and pomalidomide overcomes

bortezomib resistance, we used previously characterized(Kuhn, et al 2009) bortezomib-

sensitive (ANBL-6.WT) and -resistant (ANBL-6.BR) MM cell lines. We found significant

anti-MM activity of marizomib plus pomalidomide against ANBL-6.BR, confirming the

ability of this regimen to overcome bortezomib-resistance. Importantly, we observed similar

responses to marizomib plus pomalidomide in patient MM cells resistant to therapies

including bortezomib, lenalidomide or Dex. Moreover, the combination of marizomib and

pomalidomide overcomes the MM cell growth advantage conferred by the MM-host BM

microenvironment.

Mechanistic analysis showed that anti-MM activity of marizomib plus pomalidomide is

associated with the activation of the caspase cascade, inhibition of proteasomal activities,

and downregulation of both CRBN/IRF4/MYC signalling axis and MCL1. Studies using

biochemical inhibitors showed that blockade of either caspase-8 or caspase-9 led to a similar

degree of inhibition of marizomib plus pomalidomide-induced apoptosis. Of note, our earlier

study showed that the combination of marizomib with the immunomodulatory drug

lenalidomide relies more on caspase-8 activity than caspase-9 for inducing cell

death(Chauhan, et al 2010). Nonetheless, our data suggest that marizomib plus

pomalidomide-induced MM cell apoptosis is dependent, at least in part, on both caspase-8-

and caspase-9-mediated signalling pathways.

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Previous studies showed that marizomib inhibits CT-L, C-L and T-L proteasome activities.

(Chauhan, et al 2008b, Chauhan, et al 2005a) Our prior study also showed that lenalidomide

enhances marizomib-induced inhibition of proteasome function(Chauhan, et al 2010). In an

analogous manner, we found that the combined low concentrations of marizomib and

pomalidomide block all three proteasomal activities versus either agent alone at these low

concentrations. The mechanism whereby pomolidamide enhances the ability of marizomib

to potently inhibit proteasome activity remains to be defined. Given the immunomodulatory

characteristics of pomalidomide, it is likely that pomalidomide affects immunoproteasome

activities (CT-Li, C-Li and T-Li), which in turn may alter function of constitutive

proteasome activities. This notion is supported by earlier findings that proteasome active

sites allosterically regulate each other: for example, occupancy of C-L sites triggers the T-L

activity of proteasomes in a compensatory manner.(Kisselev, et al 2003) Indeed, additional

loss-of-function studies are required to delineate the functional role of each proteasome

subunit.

Another possible mechanism mediating the marizomib plus pomalidomide-induced

blockade of proteasome activity is the involvement of E3 ligase CRBN within the ubiquitin

proteasome pathway. CRBN is a direct protein target of pomalidomide.(Ito, et al 2010, Lu,

et al 2014) A recent study showed that CRBN modulates proteasome activity by binding to

the 20S core proteasome subunit beta type 4.(Lee, et al 2012) It is therefore possible that

CRBN may similarly affect the β5, β2 or β1 proteasome subunits to modulate overall

proteasome activity. Our genetic studies using CRBN-siRNA suggest that marizomib plus

pomalidomide-induced cell death is mediated, at least in part, by CRBN. A further evidence

for the involvement of CRBN is noted by a marked decrease in CRBN associated signalling

via IRF4 and MYC. Together, these data suggest that marizomib plus pomalidomide-

induced apoptosis is associated with a marked inhibition of all three proteasome activities,

as well as CRBN-associated downstream signalling via IRF4/MYC.

In addition to activating pro-apoptotic signaling, the combination of marizomib and

pomalidomide also downregulates proteins associated with survival and drug resistance. For

example, MCL1 is a pro-survival member of the BCL2 family, which plays a crucial role in

MM pathogenesis and drug resistance.(Fan, et al 2014) We found that low concentrations of

marizomib plus pomalidomide reduced the levels of anti-apoptotic protein MCL1. Similarly,

prior studies have linked bortezomib-induced apoptosis with the upregulation of heat shock

proteins, which confer drug resistance in MM.(Chauhan, et al 2005b, Mitsiades, et al 2002,

Obeng, et al 2006) In this context, our data shows that marizomib plus pomalidomide

combination therapy does not induce HSP90AA1, HSPA1A or HSPB1. These data suggest

that drug resistance may be less frequent in patients receiving this combined low dose

regimen.

In vitro data showing anti-MM activity of marizomib plus pomalidomide was validated in

vivo using the human MM.1S xenograft mouse model. A marked reduction in tumour

progression and prolongation of survival was observed in marizomib plus pomalidomide-

treated mice versus mice receiving either agent alone. Moreover, marizomib plus

pomalidomide treatment was well tolerated, because differences in body weight and overall

appearance were not observed. The remarkable anti-MM and anti-angiogenic activity of

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marizomib plus pomalidomide in vivo was confirmed by IHC analysis of tumours harvested

from control- and combination-treated mice using molecular markers of apoptosis

(caspase-3 cleavage, TUNEL staining), proliferation (Ki67), and associated angiogenesis

(Factor VIII and VEGFR1). Tumour lysates showed decreased MCL1 and IRF4 levels after

marizomib plus pomalidomide treatment. Therefore, these findings indicate a dual effect of

combining marizomib plus pomalidomide: increased apoptosis and decreased MM-cell

proliferation.

Collectively, our preclinical studies demonstrate potent in vitro and in vivo anti-MM activity

of marizomib plus pomalidomide at low doses that are well tolerated in a human

plasmacytoma xenograft mouse model. These findings provide the rationale for the

continuation of clinical trials of low-dose combination marizomib plus pomalidomide

therapy to enhance cytotoxicity, overcome drug resistance, reduce side effects, and improve

patient outcome in MM.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

Grant Support: This investigation was supported by National Institutes of Health Specialized Programs of Research Excellence (SPORE) grant P50100707, PO1-CA078378, and RO1 CA050947. K.C.A. is an American Cancer Society Clinical Research Professor.

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Key points

1. Combining low concentrations of marizomib and pomalidomide triggers

synergistic cytotoxicity in MM cells and overcomes bortezomib-resistance.

2. Combination of marizomib with pomalidomide represents a promising novel

therapy in patients with relapsed and refractory MM.

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Figure 1. Combination of low doses of marizomib and pomalidomide induces synergistic cell death in MM cell lines, patient MM cells but not normal PBMCs(A–D) MM-cell lines (MM.1S, ANBL6.BR, ARP-1, and MM.1R) were pretreated with or

without pomalidomide (POM) for 24 h; marizomib was then added for an additional 24 h,

followed by assessment for cell viability using the MTT assay. The experiments with single

agents and respective combinations were carried out simultaneously. Isobologram analysis

shows the synergistic cytotoxic effect of marizomib and pomalidomide. The graph (right

panel) is derived from the values given in the table (left panel). A combination index (CI) <1

indicates synergy. (E) Purified patient MM cells (CD138-positive) were pretreated with

pomalidomide for 24 h; marizomib was then added for an additional 24 h, followed by cell

death analysis using CellTiter-Glo assay. Data are mean ± SE of triplicate samples (p < 0.05

for all patient samples, for single agent vs combination treated samples). (F) Peripheral

blood mononuclear cells (PBMCs) from healthy donors were treated (as in panel A) with

indicated concentrations of marizomib, pomalidomide or marizomib plus pomalidomide,

and then analysed for viability using the WST-1 assay. Data are mean ± SE (n=5). Fa =

fraction of cells affected.

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Figure 2. Combined low doses of marizomib and pomalidomide block migration, tubule formation, and cytoprotective effects of BMSCs and pDCs(A) Migration assay: MM.1S cells were pretreated with pomalidomide for 12h, and then

marizomib was added for an additional 6h; cells were more than 90% viable at this time

point. The cells were washed and cultured in serum-free medium. After 2h incubation, cells

were plated on a fibronectin-coated polycarbonate membrane in the upper chamber of

transwell inserts and exposed for 4h to serum-containing medium in the lower chamber.

Cells migrating to the bottom face of the membrane were fixed with 90% ethanol and

stained with crystal violet. A total of 3 randomly selected fields were examined for cells that

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had migrated from the top to the bottom chambers. (Left panel) Image is representative of 2

experiments with similar results. (Right panel) The bar graph represents quantification of

migrated cells. Data are mean ± SE (p < 0.05). (B) Human vascular endothelial cells were

cultured in the presence or absence of combined low doses of marizomib plus pomalidomide

for 48 h, and then assessed for in vitro angiogenesis using matrigel capillary-like tube

structure formation assays (Left panel). Image is representative from 3 experiments with

similar results. In vitro angiogenesis is reflected by capillary tube branch formation (dark

brown). (Right panel) The bar graph represents quantification of capillary-like tube structure

formation in response to indicated agents: Branch points in several random view fields/well

were counted, values were averaged, and statistically significant differences were measured

using Student’s t test. (C–D) MM.1S cells were cultured in bone marrow stromal cells

(BMSC)- or plasmacytoid dendritic cells (pDC)-coated or uncoated wells with control

medium, marizomib, pomalidomide or marizomib plus pomalidomide. Cell proliferation

was assessed by Brdu colorimetric assay. Data are mean ± SE (n=3; p < 0.05, for control vs

combination treated samples).

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Figure 3. Marizomib plus pomalidomide-induced apoptosis in MM cells is associated with activation of caspases(A) MM.1S, RPMI-8226 or Dox-40 cells were pre-treated with or without pomalidomide for

24 h, and then marizomib was added for an additional 24 h; protein lysates were subjected to

immunoblot analysis with anti-PARP, anti-caspase-3, anti-caspase-8, anti-caspase-9 or anti-

GAPDH FL, full length; CF, cleaved fragment. Blots shown are representative of 3

independent experiments. (B) MM.1S cells were pre-treated with or without pomalidomide

for 24 h, and then marizomib was added for an additional 24 h; protein lysates were

subjected to measurement of caspase-8 or caspase-9 enzymatic activity using fluorometric

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kit. Data are mean ± SE (n=3; p < 0.05, for single agent vs combination treated samples) (C)

MM.1S cells were treated with indicated agents (as in panel ‘A’) in the presence or absence

of biochemical inhibitors of caspase-3, caspase-8, caspase-9, or pan-caspase and then

analyzed for apoptosis using WST-1 assay. Data are mean ± SE (n=3; p < 0.05, for control

vs combination treated samples).

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Figure 4. Mechanisms mediating anti-MM activity of marizomib plus pomalidomide(A) MM.1S cells were pretreated with or without pomalidomide for 12 h, and then

marizomib was added for an additional 12 h; cells were harvested and cytosolic extracts

were then analysed for chymotrypsin- like (CT-L), caspase-like (C-L) and trypsin-like (T-L)

proteasome activities. Results are represented as percentage inhibition in proteasome

activities in drug-treated versus vehicle control. Data are mean ± SE (n=3; p < 0.05, for

single agent vs combination treated samples) (B) Total protein lysates from the indicated

MM cell lines and normal healthy donor PBMCs were subjected to immunoblot analysis

with anti-CRBN or anti-GAPDH Abs. (C) MM.1S cells were transfected with siRNA-

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CRBN or scr-siRNA for 24h. Transfected cells were pretreated with or without

pomalidomide for 24h; marizomib was then added for an additional 24h, followed by

analysis for apoptosis using WST-1 assay. Data are mean ± SE (n=3; p < 0.05, for single

agent vs combination treated samples) Immunoblot shows CRBN expression in cells

transfected with scr-siRNA or CRBN-siRNA. (D and E) MM.1S cells were treated with

indicated agents (as treated in panel ‘A’); protein lysates were subjected to immunoblot

analysis with anti-CRBN, anti-IRF4, anti-MYC, anti-MCL1, anti-HSP90AA1, anti-

HSPA1A, anti-HSPB1 or anti-GAPDH Abs.

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Figure 5. Combination of low doses of marizomib and pomalidomide inhibits human plasmacytoma growth in CB-17 SCID mice(A) Mice bearing human MM.1S MM tumours (n=5/group) were treated with vehicle,

marizomib (0.15 mg/kg; orally), pomalidomide (0.5 mg/kg or 2.5 mg/kg; orally), or

marizomib plus pomalidomide (orally) at the indicated doses for 24 days on a twice-weekly

schedule for marizomib and for 4 consecutive days weekly for pomalidomide. Bars

represent mean ± SE. (B) Kaplan-Meier plots showing survival for mice treated with

marizomib, pomalidomide, or marizomib plus pomalidomide at the indicated doses.

Marizomib plus pomalidomide-treated mice show significantly increased survival (p < 0.05,

Logrank test for trend) compared with the untreated group. (C) Vehicle-treated control mice,

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as well as mice in the marizomib, pomalidomide, or marizomib plus pomalidomide-treated

cohorts, were weighed every week. The average changes in body weight are shown. (D)

Mice were treated with vehicle, marizomib, pomalidomide, or marizomib plus

pomalidomide (as in panel ‘A’) for 24 days; blood samples were then obtained and

subjected to analysis for serum bilirubin, hemoglobin, and creatinine levels using

Quantichrom Creatinine, Bilirubin, and Haemoglobin Assay kit (BioAssay Systems,

Hayward, CA, USA).

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Figure 6. Effect of marizomib plus pomalidomide on apoptosis, MCL1, IRF4 and neovascularization in vivo in xenografted MM tumours(A and B) Apoptotic cells in sections of tumours harvested from vehicle control-,

marizomib-, pomalidomide-, or marizomib (0.15 mg/kg) plus pomalidomide (2.5 mg/kg)-

treated mice were identified by immunostaining for activated caspase-3 (green cells) or

TUNEL. Tumour sections were obtained on day 24. Images were obtained with a Leica

SP5X laser scanning confocal microscope (40x magnification). (C) Tumours harvested from

mice (as in panel ‘A’) were immunostained with Ki67 Abs (40x magnification). (D) Tumour

lysates from control and drug-treated mice were subjected to immunoblot analysis using

anti-MCL1, anti-IRF4, or anti-GAPDH Abs. Lanes 1 to 6 represent lysates of tumours

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harvested from mice receiving the following treatments: lane 1, vehicle alone (control); lane

2, marizomib (0.15 mg/kg); lane 3, pomalidomide (0.5 mg/kg); lane 4, pomalidomide (2.5

mg/kg); lane 5, marizomib (0.15 mg/kg) plus pomalidomide (2.5 mg/kg); and lane 6,

marizomib (0.15 mg/kg) plus pomalidomide (0.5mg/kg). (E and F) Tumours harvested from mice (as in panel ‘A’) were immunostained with Factor VIII or VEGFR1 Abs. Images

were obtained with a Leica SP5X laser scanning confocal microscope (40x magnification).

Photographs (A, B, C, E, and F) shown are representative of similar observations in 2 mice

receiving the same treatment.

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