The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cells in...

The investigational agent MLN2238 induces apoptosis and iscytotoxic to CLL cells in vitro, as a single agent and incombination with other drugs

Aneel Paulus,1,2* Aisha Masood,3,4* Kena

C. Miller,2 A. N. M. Nazmul H. Khan,3,5

Drusilla Akhtar,3 Pooja Advani,2 James

Foran,2 Candido Rivera,2 Vivek Roy,2

Gerardo Colon-Otero,2 Kasyapa Chitta1

and Asher Chanan-Khan2

1Department of Cancer Biology, Mayo Clinic,

Jacksonville, FL, 2Division of Hematology and

Oncology, Mayo Clinic, Jacksonville, FL,3Department of Medicine, Roswell Park Cancer

Institute, Buffalo, NY, 4Department of Internal

Medicine, State University of New York at Stony

Brook University, Stony Brook, NY, and5Department of Infectious Diseases, Roswell Park

Cancer Institute, Buffalo, NY, USA

Received 17 July 2013; accepted for publication

13 October 2013

Correspondence: Dr Asher Chanan-Khan,

Division of Hematology & Oncology, Mayo

Clinic Cancer Center, 4500 San Pablo Road,

Jacksonville, FL 32224, USA.

E-mail: [email protected]

*Contributed equally to the study.

Summary

Chronic lymphocytic leukaemia (CLL) is the most common haematological

malignancy in the U.S. The course of the disease has been shown to be

negatively impacted by increased levels of BCL2. Strategies to downregulate

BCL2 and shift the balance towards cellular demise are actively being

explored. Therefore, we examined whether the investigational agent

MLN2238 could inhibit the proteasomal machinery and induce CLL cell

death while also downregulating BCL2. MLN2238-induced cell death was

studied in peripheral blood mononuclear cells from 28 CLL patients.

MLN2238 produced a dose-dependent reduction in BCL2 and CLL cell via-

bility with maximum cell death observed at a 50 nmol/l concentration by

48 h. Annexin-V staining, PARP1 and caspase-3 cleavage along with an

increase in mitochondrial membrane permeability were noted after cells

were treated with MLN2238; however, apoptosis was only partially blocked

by the pan-caspase inhibitor z-VAD.fmk. Furthermore, we observed

enhanced anti-CLL effects in tumour cells treated with either a combina-

tion of MLN2238 and the BH3 mimetic AT-101 or MLN2238 and fludara-

bine. Together, our data suggest the potential for proteasome inhibitor

based therapy in CLL and the rationale design of drug combination strate-

gies based on CLL biology.

Keywords: MLN2238, MLN9708, Ixazomib, CLL, AT-101.

Chronic lymphocytic leukaemia (CLL) is the most common

leukaemia in the western hemisphere.(Dores et al, 2007) CLL

manifests as a clinically heterogeneous cancer as some

patients never require therapy, while those with prominent

genetic defects respond poorly to standard chemoimmuno-

therapeutic agents and often develop relapsed/refractory dis-

ease (Rai et al, 1975; Hallek & Pflug, 2010; Wierda et al,

2010; Advani et al, 2011). In order to improve disease

outcome, the accurate identification and rational targeting

of pro- and anti-apoptotic signalling pathways is crucial,

remaining a priority of translational research in CLL.

Defects in apoptosis are known to result in aggressive dis-

ease behaviour and confer a poor prognosis in most cancers,

including CLL (Reed et al, 2002). Importantly, a shift between

the pro-apoptotic and anti-apoptotic BCL2 family of proteins

is associated with a dysfunctional programmed CLL cell death

response (Schena et al, 1993; Thomas et al, 1996; Reed, 1997).

The underlying mechanisms that influence the expression of

BCL2 proteins are complex and remain an investigational

priority for therapeutic exploit. The ubiquitin-proteasome

pathway is an integral system involved in governing BCL2

family members. This pathway also controls the degradation

of various intracellular proteins (Fennell et al, 2008). Its

central component, the proteasome, is responsible for the

recycling and destruction of transcription factors, such as

TP53 (p53) and NFKB1 (nuclear factor-jB, NF-jB), and

cyclin-dependent kinase inhibitors (Adams, 2003). The ubiqu-

itin-proteasome pathway also plays an important role in the

regulation and stabilization of pro-apoptotic molecules (par-

ticularly the BH3 domain proteins BIK, PMAIP1 [NOXA]

and BCL2L11 [BIM]) and as such, links the apoptotic

machinery to the protein disposal systems of the proteasome.

Inhibition of this pathway via proteasome inhibitors (PIs) has

been shown to cause accumulation of TP53, p27 and pro-

apoptotic BCL2 proteins, which results in activation of the

mitochondria- mediated cell death pathway (Fennell et al,

2008). Thus, identification and correction of defects that affect

apoptosis may offer a therapeutic opportunity to reset and

research paper

First published online 27 January 2014doi: 10.1111/bjh.12731

ª 2014 John Wiley & Sons LtdBritish Journal of Haematology, 2014, 165, 78–88

engage cell death pathways in CLL. Bortezomib (VELCADE�,

Millennium Pharmaceuticals, Inc., The Takeda Oncology

Company,Cambridge, MA, USA) was the first in class PI and

was the first PI to be approved for the treatment of multiple

myeloma (MM) and relapsed mantle cell lymphoma. As inves-

tigated in CLL cells in a preclinical setting, cytotoxic activity

of bortezomib is associated with alteration of mitochondrial

outer membrane permeability (MOMP) and caspase activa-

tion along with up regulation of PMAIP1 and BBC3 [PUMA]

in vitro (Pahler et al, 2003; Pei et al, 2003). However, this bio-

logical activity did not wholly translate into an observable

clinical benefit when the efficacy of single agent bortezomib

was examined in fludarabine-refractory CLL patients. This

was speculated to be a result of the single agent use of bort-

ezomib in a heavily pretreated patient population (median

lines of prior therapy = four, range 2–11)(Faderl et al, 2006).

Moreover, the presence of flavonoids, such as quercetin and

myricetin, in the plasma have been attributed to the preven-

tion of CLL cell death and are linked to the chemical reaction

between quercetin and the boronic acid group in bortezomib

(Liu et al, 2008; Wickremasinghe, 2008). Thus, PIs that do

not contain a boron moiety and the partnership of these com-

pounds with additional drugs whose activity complements

theirs may be necessary to achieve clinical remission in heavily

pretreated CLL patients. As such, we sought to determine the

anti-leukaemic effects of the investigational PI MLN2238 (ix-

azomib), which has different pharmacokinetic and structural

properties to bortezomib. The investigational drug MLN9708

(ixazomib citrate), which is an orally available small molecule

PI, converts into the biologically active form, MLN2238, upon

hydrolysis. Compared with bortezomib in preclinical models,

MLN2238 demonstrates a faster dissociation rate from the

proteasome and improved pharmacokinetic, pharmacody-

namic and antitumour properties in preclinical models

(Kupperman et al, 2010; Chauhan et al, 2011). Pre-clinical

data suggest a synergistic effect when PIs are coupled with

BCL2 inhibitors (Pei et al, 2003; Perez-Galan et al, 2007),

prompting us to explore this combination with MLN2238.

In the current study, we examined the anti-CLL effect of

MLN2238. We aimed to identify the best combination-based

therapeutic strategy in vitro using CLL patient cells. We

found that treatment with MLN2238 leads to induction of

apoptosis in primary CLL cells and, when combined with the

pan-BCL2 inhibitor AT-101, results in increased tumour kill.

Similarly, the combination of MLN2238 plus conventional

cytotoxic agents (fludarabine or dexamethasone) also resulted

in increased CLL cell death as compared to when each agent

was used alone.

Materials and methods

Patients

Chronic lymphocytic leukaemia cells were obtained from

patients with a confirmed diagnosis of CLL. All patients

provided written informed consent to participate in the

study. This study and the consent form were approved by

the Roswell Park Cancer Institute institutional review board

in accordance with the Declaration of Helsinki. Only patients

with a high total white blood cell count in the peripheral

blood that also had more than a 90% CD19+ B cell popula-

tion (hereafter referred to as CLL cells) were included in this

study.

Cell isolation, culture and drug treatment

Heparinized peripheral blood was obtained from patients

(n = 29) with CLL. PBMCs were separated on a Ficoll gradi-

ent, washed twice in phosphate-buffered saline (PBS), and

resuspended in culture medium (RPMI-1640 containing 10%

fetal bovine serum [FBS] and 1% penicillin-streptomycin).

Cell viability was determined using a Vi-Cell-XR cell viability

ysanalyser (Beckman Coulter, Brea, CA, USA). Experiments

were done with cell concentrations of 5 9 106/ml. MLN2238

was from Millennium Pharmaceuticals Inc. (Cambridge, MA,

USA), AT-101 was provided as a gift from Ascenta Thera-

peutics Inc. (Malvern, PA, USA), fludarabine and lenalido-

mide were purchased from Sellekhem (Houston, TX, USA),

and dexamethasone was purchased from Sigma–Aldrich

(St. Louis, MO, USA).

Proteasomal activity assay

Proteasomal activity was determined by using synthetic fluor-

ogenic peptide substrates. Briefly, the cells were washed twice

with cold PBS and the total cell extracts were made in lysis

buffer containing 25 mmol/l HEPES, pH 7�5, 500 lmol/l

EDTA, 0�05% Nonidet P-40, and 0�001% sodium dodecyl

sulfate (SDS) (w/v) without protease inhibitors, to a final

concentration of 4 9 106 cells/ml. The reaction mixture con-

taining 10 ll of the lysate (40 000 cells) was incubated at

37°C for 30 min with the PI, followed by the addition of

50 lmol/l fluorogenic peptides (Suc-Leu-Leu-Val-Tyr-AMC

(LLVY; for chymotrypsin-like activity), Ac- Leu-Arg-Arg-

AMC (LRR; for tryspin-like activity), or Z-Leu-Leu-Glu-

AMC (LLE; caspase-like activity) and further incubated for

60 min at 37°C. Release of the fluorometric reporter, ami-

nomethylcoumarin (AMC) as a result of the activity of the

respective enzymes was quantified in a BioTek FL800 plate

reader using 360 nmol/l excitation and 460 nmol/l emission

wavelengths. Enzymatic activity represents the mean fluores-

cence values of triplicate independent assays.

Apoptosis assay

Apoptosis was measured by the annexin V binding assay kit

from Pharmingen (San Diego, CA, USA) according to manu-

facturer’s instructions. Briefly, cells were washed with PBS

and 1 9 106 cells were resuspended in 100 ll of 1 9 binding

buffer. Fluorescein isothiocyanate (FITC)-labelled Annexin

MLN2238 Induces Apoptosis and is Cytotoxic to CLL Cells

ª 2014 John Wiley & Sons Ltd 79British Journal of Haematology, 2014, 165, 78–88

V (5 ll) and propidium iodide (10 ll) were added to each

sample and incubated in the dark for 15 min at room tem-

perature. Subsequently, cells were analysed by flow cytome-

try. Data from 10 000 events per sample were collected and

processed using CELL QUEST software (Becton Dickinson,

Franklin Lakes, NJ, USA).

Determination of Mitochondrial Outer MembranePermeability (MOMP)

Chronic lymphocytic leukaemia cells treated with MLN2238

were tested for MOMP using tetramethylrhodamine methyl

ester [TMRM] (Invitrogen, Carlsbad, CA, USA). The cells

were washed twice with PBS, incubated in PBS containing

20 nmol/l TMRM for 15 min and analysed for fluorescence

on a FACScaliber flow cytometer (FL2). Data from at least

10 000 events per sample were collected and analysed using

the CELL QUEST software (Becton Dickinson). TMRM-negative

(%) cells were calculated to determine (%) MOMP.

Immunoblot analysis

Total protein extracts were made using radioimmunoprecipi-

tation assay (RIPA) lysis buffer (50 mmol/l Tris containing

150 mmol/l NaCl, 0�1% SDS, 1% TritonX-100, 1% sodium

deoxycholate, pH 7�2) with 0�2% protease and phosphatase

inhibitor cocktail (Sigma, St. Louis, MO, USA) on ice for

40 min, vortexing for 5 s every 10 min. Following centrifuga-

tion at 18 400 g for 20 min, the supernatant was collected

and used for Western blot analyses. Protein content in the

extracts was measured by the Bradford method using Bio-

Rad protein assay reagent (Bio-Rad Laboratories, Hercules,

CA, USA). Aliquots of total protein (30 lg) and 25 lg of

nuclear/cytoplasmic protein were boiled in Laemmli sample

buffer and subjected to 10% SDS-polyacrylamide gel electo-

phoresis (SDS-PAGE) and transferred onto a polyvinylidene

difluoride membrane. Membranes were blocked for 1 h in

Tris-buffered saline/Tween 20 [TTBS] containing 1% nonfat

dried milk and 1% BSA. Incubation with primary antibodies

was done overnight at 4°C, followed by washing three times

with TTBS and incubation for 1 h with horseradish peroxi-

dase-conjugated secondary antibody. The blots were devel-

oped using chemiluminescence (Thermo Scientific, Waltham,

MA, USA).

Results

MLN2238 inhibits proteasomal activity in CLL cells

MLN2238 is the biologically active form of the investiga-

tional proteasome inhibitor MLN9708. It has a shorter

proteasome dissociation half-life and improved pharmacoki-

netics and pharmacodynamics compared to bortezomib in

preclinical models (Kupperman et al, 2010). We conducted

in vitro studies to understand the effect of MLN2238 in CLL

cells. Proteasomal activity of CLL cells was determined as

described in materials and methods using fluorogenic peptide

substrates. Variable basal activity was detected in CLL cells

from all patients investigated for the three major catalytic

components of the proteasome that confer chymotrypsin-

like, trypsin-like and caspase-like enzymes. Chymotrypsin-

like activity from eight representative patient samples is

shown in Fig 1A. The effect of MLN2238 on proteasomal

(A)

(B)

(C)

Fig 1. Chymotrypsin-like activity in the proteasomes of B-CLL cells

is inhibited by MLN2238 in vitro: Chymotrypsin-like, caspase-like

and tryspin-like activities (using their respective fluorogenic peptides

as described in Materials and methods) were measured in primary

B-CLL cells at 4 9 104 cells/reaction in triplicates. (A) Chymotryp-

sin-like activity from eight representative patients samples shows var-

iation in baseline activity. (B) Chymotrypsin-like activity in the cells

was most significantly inhibited (P < 0�005) by 10 nmol/l MLN2238

in all samples tested (n = 28). Data from four patients is presented.

(C) Western blot analysis of protein extracts from Chronic lympho-

cytic leukaemia (CLL) cells treated with 10 nmol/l MLN2238 for

24 h for the presence of PSMB5 showed that PSMB5 protein levels

are not altered in in presence of MLN2238. b-actin was used as con-

trol for equal protein loading.

A. Paulus et al

80 ª 2014 John Wiley & Sons LtdBritish Journal of Haematology, 2014, 165, 78–88

enzyme activity was measured by pre-incubating the cell

extracts with 10 nmol/l of MLN2238 for 30 min followed by

incubation with specific fluorogenic substrate for another

60 min. MLN2238 inhibited the chymotrypsin-like activity

by more than 90% (P < 0�005) (Fig 1B). A moderate to min-

imal inhibitory effect on caspase-like and trypsin-like activi-

ties, respectively, was also noted (data not shown). As

chymotrypsin-like activity is mediated by the b5 subunit of

the proteasome (PSMB5 gene), the effect of MLN2238 on

PSMB5 protein levels was evaluated by Western blot analysis.

PSMB5, a 23 kDa protein, was detectable in CLL cells show-

ing measurable chymotrypsin-like activity (data from three

representative patients is presented in Fig 1C) and was not

detectable in patients with low levels of enzyme activity (one

sample from Fig 1C), which suggests a low threshold expres-

sion in these cells. Further, treatment of CLL cells for 24 h

with 25 nmol/l MLN2238 did not inhibit PSMB5 protein

levels, which suggests that MLN2238 inhibits the catalytic

activity of the proteasome without changing PSMB5 protein

levels. Collectively, these results indicate that MLN2238

potently inhibits chymotrypsin-like proteasomal activity in

CLL cells without affecting protein expression levels of

PSMB5.

Exposure to MLN2238 results in the loss of cell viabilityand induces apoptosis in CLL cells

Sustained proteasomal activity is indispensable for cellular

protein homeostasis, viable maintenance and growth. Thus,

we hypothesized that CLL cells are dependent on proteaso-

mal activity for their survival and that MLN2238 treatment

would induce death in CLL cells. CLL cells from 18 represen-

tative patients were treated with different concentrations of

MLN2238 for 24 h and cell viability was determined at 48 h

by the trypan blue exclusion assay. Cells showed a concentra-

tion-dependent decrease in viability with 42% cell death

noted at a 50 nmol/l concentration of MLN2238 (Fig 2A).

Next, we sought to delineate the mechanism of death that

occurred in response to MLN2238 exposure via staining with

annexin V and propidium iodide. MLN2238-treated CLL

cells underwent apoptosis in a dose-dependent manner with

a maximal effect at a 50 nmol/l concentration (Fig 2B). Cell

death was observed in a median of 43% of cells at 25 nmol/l

(range 10–54%) and 60% cells at 50 nmol/l (range,

25–73%). Apoptotic effects of MLN2238 were not associated

with clinical stage of the disease or number of previous treat-

ments that the patients had been exposed to. Additionally,

status of ZAP70, immunoglobulin expression, IGHV muta-

tion status, TP53 expression, ATM mutation or chromo-

somal trisomy did not alter the extent of apoptosis induced

by MLN2238 (Table I). Induction of apoptosis was

confirmed by PARP cleavage, which occurred in a dose-

dependent manner (Fig 2C). The results indicated that

MLN2238 induces a dose-dependent induction of apoptosis

in CLL cells.

MLN2238 activates caspases 3 and 9 in CLL cells;however, apoptosis is caspase independent

To determine if MLN2238-induced apoptosis in CLL is cas-

pase-mediated, CLL cells were treated with increasing con-

centrations of MLN2238 for 24 h and the protein extracts

were probed for caspases 9, 8 and 3. While untreated cells

showed only the full length forms of caspase 9 and 3 at

45 kDa and 35 kDa respectively, treatment with MLN2238

(A)

(B)

(C)

Fig 2. Loss of Chronic lymphocytic leukaemia (CLL) cell viability

and induction of apoptosis in primary CLL cells by MLN2238 in

vitro: (A) B-CLL cells from 18 representative patients were treated

with 12�5, 25, 50 nmol/l concentrations of MLN2238 for 24 h and

cell viability was determined at 48 h. Cells showed a concentration-

dependent decrease in viability with 58% of cells remaining viable at

a concentration of 50 nmol/l. (B) Apoptosis was assessed by FITC

labelled annexin V (5 ll) and propidium iodide. Subsequently, sam-

ples were analysed by flow cytometry. Maximum apoptosis occurred

at 50 nmol/l of MLN2238 at 24 h. (C) B-CLL cells from Patient 4

were incubated with 0, 25, 50 and 100 nmol/l of MLN2238 for 24 h

and induction of PARP1 cleavage was measured by Western blot.

ACTB (b-actin) was used as a control.



resulted in an increased appearance of small cleaved pro-

teins in a dose-dependent manner (at 35 kDa for caspase 9

and 19 kDa for caspase 3) indicating activation of these

two caspases and the intrinsic apoptotic pathway (Fig 3A).

Activation of caspase 8 was not observed in CLL cells trea-

ted with MLN2238 [data not shown]. To evaluate the

dependence of MLN2238-mediated apoptosis on the activa-

tion of caspases, CLL cells were pre-treated for 1 h with

25 lmol/l z-VAD.fmk, a pan caspase inhibitor, followed by

25 nmol/l MLN2238 for 24 h and the extent of apoptosis

was ascertained. Treatment with z-VAD.fmk alone did not

induce the activation of caspase-3, which was similar to

untreated cells. While MLN2238 induced the activation of

caspase-3 in these cells, pre-treatment with z-VAD.fmk

blocked this activation, which suggests the inhibition of cas-

pase activation in these cells in presence of z-VAD.fmk

(Fig 3B). However, cell death by MLN2238, as determined

by annexin V staining, was not abrogated by pre-treatment

with z-VAD.fmk (Fig 3C). This observation suggests that

MLN2238-mediated apoptosis in CLL cells can occur inde-

pendently of caspase activation, despite their induction in

the presence of the drug.

MLN2238 induces MOMP in CLL cells

Activation of caspases 9 and 3 in CLL cells treated with

MLN2238 indicated a possible involvement of the mitochon-

drial-mediated intrinsic apoptosis pathway. To test our

hypothesis, we investigated MOMP in CLL cells treated with

MLN2238. CLL cells were incubated alone or with increasing

concentrations of MLN2238 for 24 h. MOMP was measured

using TMRM by flow cytometry as described in the materials

and methods section. Untreated cells that tested positive for

TMRM fluorescence became progressively negative for TMRM

with increasing concentrations of MLN2238, which suggests

that that MLN2238 treatment increased MOMP in a dose-

dependent manner. All the samples used in the study showed

an increase in MOMP. Data from eight representative patients

is presented in Fig 3D.

BCL2 expression is decreased in MLN2238 treated CLLcells

One of the possible mechanisms of increase in MOMP in

CLL cells treated with MLN2238 could be due to a shift in

Table I. Clinical characteristics of patients whose cells were used in study (n = 28).

Pt. Age (years) Stage ALC (9 10 (/l) Prior therapies (n) IGHV status b2M 17p 11q

1 41 NA 48�63 0 NA 1�61 NA Normal

2 84 IV NA 2 NA 1�93 Normal Normal

3 63 NA 20�62 2 NA 3�32 Normal Normal

4 52 II 71�37 0 NA 3�75 Normal Deletion

5 40 NA 20�67 0 Mutated 2�91 Normal Normal

6 63 0 36�8 0 Mutated 1�71 Normal Normal

7 63 NA 20�62 2 NA 3�32 Normal Normal

8 70 IV 10�35 3 NA 4�71 Normal NA

9 65 I NA NA NA NA NA Normal

10 56 II NA 1 NA 3�93 Normal Normal

11 54 I 16�24 1 NA 1�8 Normal Deletion

12 82 NA 94�64 4 NA 6�53 Deletion Deletion

13 75 IV 1�93 1 NA 8�43 Normal Normal

14 66 I 44�29 1 NA 3�24 Normal NA

15 64 IV 0�53 1 Mutated 5�42 NA Deletion

16 59 IV 3�73 8 NA 3�48 Normal Deletion

17 69 IV NA 4 NA 5�79 Deletion Normal

18 67 IV NA 5 NA 2�45 Normal Deletion

19 72 I 0�93 1 NA 2�16 Normal Normal

20 66 IV NA 4 NA 3�48 Normal Deletion

21 60 NA NA NA Mutated 2�01 Normal Normal

22 62 IV 40�58 0 Mutated 4�97 Normal Normal

23 66 II NA 1 NA 6�09 Normal Deletion

24 65 IV NA 1 NA 2�93 Normal Normal

25 87 0 34�95 0 Unmutated 7�84 Normal Normal

26 81 IV 3�81 2 NA 5�05 Normal Deletion

27 66 I 2�28 1 Unmutated 3�49 Normal Normal

28 61 I 51�29 0 NA 1�39 Normal Normal

29 68 II 0�55 4 Unmutated 4�52 Normal Normal

ALC, absolute lymphocyte count; NA, not available; IGHV, immunoglobulin heavy chain mutation status; b2M, beta 2 microglobulin; Pt., patient;

17p, chromosome 17p; 11q, chromosome 11q.

A. Paulus et al


the balance between pro-and anti-apoptotic proteins of the

BCL2 family. To evaluate this hypothesis, CLL cells were

treated with increasing concentrations of MLN2238 for 24 h

and the protein extracts were analysed for the presence of

BCL2 by Western blot analysis. MLN2238-treated cells exhib-

ited reduced expression of pro-survival BCL2 members as

compared to untreated CLL cells (data from two representa-

tive patients, Fig 4A). Inhibition of BCL2 was observed as

early as 12 h after treatment with MLN2238, which suggests

that altered BCL2 expression is one of the early events associ-

ated with MLN2238-mediated apoptosis of CLL B-cells.

These results suggest that the apoptosis-inducing function of

MLN2238 is mediated in part by its ability to inhibit expres-

sion of the BCL2 family of pro-survival proteins.

The combination of MLN2238 and AT-101 (BH3mimetic) induces robust CLL cell death

We have previously shown that AT-101, which is a BH3

mimetic, downregulates BCL2 in WM (Chitta et al, 2009)

and primary CLL cells (Masood et al, 2011). The observation

that MLN2238 also targets BCL2 proteins suggests its poten-

tial for use in combination with BCL2 modulators. To test

this, we investigated the effect of AT-101 in combination with

MLN2238 on CLL cells. BCL2 protein expression was mea-

sured in response to MLN2238 (Fig 4A, data from two repre-

sentative patients shown), AT-101 or the combination of the

two agents together (Fig 4B). BCL2 expression level in CLL

cells was decreased by both agents alone and more notably so

in AT-101-treated cells (data from one representative patient

shown). We next investigated whether AT-101 could be suc-

cessfully combined with MLN2238 for enhanced tumour kill.

As a single agent, MLN2238 induces robust CLL cell death at

50 nmol/l (~50% loss of cell viability), ergo we rationalized

that AT-101 could augment the effects of sub-apoptotic con-

centrations of MLN2238. We treated CLL cells from three

patient samples with MLN2238 (12�5 nmol/l) plus AT-101

(2�5 lmol/l or 5 lmol/l) for 24 h. The cells were subse-

quently stained with annexin V and propidium iodide fol-

lowed by flow cytometric analysis of apoptotic cell death. We

sought to determine if AT-101 and MLN2238 could be com-

bined for enhanced CLL tumour kill. Single agents MLN2238

(12�5 nmol/l) and AT-101 (2�5 lmol/l) produced an average

of 3�23% and 9�3% cell death over control cells. As hypothe-

sized, we observed enhanced tumour cell death (16% over

control) when AT-101 (2�5 lmol/l) was combined with

MLN2238 (12�5 nmol/l), which indicated that a BCL2 inhibi-

tor could effectively lower the apoptotic threshold of CLL

cells, rendering them vulnerable to lower concentrations of

MLN2238 (Fig 4C, left bars). Apoptotic cell death at these

concentrations was also confirmed by immunoblotting for the

cleaved protein product of PARP1 (Fig 4D). Interestingly,

although greater percent cell death (average 33%) was noted

in CLL cells treated with a higher concentration (5 lmol/l) of

(A) (B)

(C) (D)

Fig 3. MLN2238 activates the intrinsic apoptotic pathway in B-CLL cells: (A) B-CLL cells were treated with 25, 50 and 100 nmol/l concentrations

of MLN2238 for 24 h with subsequent gauging of caspase cleavage by Western blot. Cleavage products of caspases 9 and 3 occured starting at

25 nmol/l in treated cells. (B) Extent of caspase 3 involvement in mediating CLL cell death was determined by pre-treated cells for 1 h with

25 lmol/l z-VAD.fmk, a pan caspase inhibitor, followed by MLN2238 (25 nmol/l) for 24 h. Treatment with z-VAD.fmk alone did not induce

activation of caspase-3. While MLN2238 induced cleavage of caspase-3 in these cells, pre-treatment with z-VAD.fmk blocked the cleavage. (C)

However, cell death by MLN2238, as determined by annexin V staining, was not abrogated by pre-treatment (2 h) with z-VAD.fmk, which sug-

gests that caspase-independent mediated cell death mechanisms are activated by MLN2238 in spite of caspase induction. (D) Cells were treated

with 25 and 50 nmol/l of MLN2238 for 24 h and mitochondrial outer membrane permeability (MOMP) activation was analysed using tetrameth-

ylrhodamine methyl ester. Maximum loss of MOMP occurred at a 50 nmol/l concentration of MLN2238. Data from eight representative patient

samples is shown.



single agent AT-101, we noticed only a marginal benefit fol-

lowing the addition of MLN2238 (Fig 4C, right bars).

MLN2238 enhances the anti-CLL activity of fludarabineor dexamethasone

MLN2238 combined with AT-101 demonstrated substantial

cytotoxic activity; thus, we investigated the effect of MLN2238

on CLL cells in combination with the standard-of-care

anti-CLL agents, fludarabine or dexamethasone, along with the

immunomodulatory drug (IMiD) lenalidomide. In these

experiments, we aimed to determine the effect of MLN2238

as an adjunct to the aforementioned chemoimmunotherapies.

Thus we used both an optimal (50 nmol/l) and sup-optimal

(25 nmol/l) concentration of MLN2238. With the addition

of lenalidomide to MLN2238 pre-treated cells, no cytotoxic

effects were observed by 24 h (data not shown). In contrast,

the addition of MLN2238 to fludarabine- (1 lmol/l) or

dexamethasone- (10 lmol/l) treated CLL cells resulted in

increased apoptotic cell death by annexin V and propidium

iodide staining. Using the lower concentration (25 nmol/l) of

MLN2238 (M) in combination with dexamethasone, we

observed no significant change in the percent tumour cell

death over that of dexamethasone alone (Fig 5A, bar D+M).

However, at a 50 nmol/l concentration (M1), MLN2238 plus

dexamethasone induced significant (P < 0�05) apoptosis in

45% of malignant tumour cells, compared to control. The

average percent tumour cell death in dexamethasone-treated

cells was 15%; in MLN2238 [50 nmol/l]-treated cells, it was

30�66% (Fig 5B, bar D+M1). The effects of the lower concen-

tration of MLN2238 were more notable in the MLN2238–

fludarabine combination, which showed increased tumour

kill at both the low (20% average tumour kill) and higher

doses of MLN2238 (45% average tumour kill) (Fig 5B).

The results suggest that MLN2238 can sensitize CLL cells

for their effective targeting by chemotherapeutic anti-CLL

drugs.

Discussion

Proteasome inhibitors are potent compounds with diverse

effects on cell signalling circuits, such as inactivation of

NFKB1, stabilization of TP53 and pro-apoptotic proteins

(BID, BAX, PMAIP1) and downstream activation of stress

related pathways (Adams, 2003). In this study, we tested

MLN2238, the biologically active form of the investigational

PI MLN9708, for its direct proteasome-targeted anti-CLL

activity. We examined its effects on the downstream auxiliary

leukaemogenic pathways that drive CLL proliferation

through derangements in the mitochondrial apoptotic sys-

tem. MLN2238 actively induced apoptosis in all CLL patient

samples irrespective of the patients’ disease stage, genomic

aberrations or number of prior therapies. Further, we dem-

onstrated that MLN2238 induced apoptosis in CLL cells by

targeting anti-apoptotic proteins of the BCL2 family, thus

offering an effective strategy to regulate the balance between

cell survival and apoptosis.

We observed that MLN2238 induces changes to MOMP,

PARP1, and caspase activation. Treating the cells with

z-VAD.fmk inhibits induction of caspases; however, cell

death is not affected. This is not entirely surprising, because

disruption of the proteasome can lead to increases in a myriad

of protein substrates that are capable of inducing cellular

apoptosis independent of caspase activation. These mecha-

nisms may include inhibition of NFKB1, activation of the

endoplasmic reticulum (ER) stress response, which is caused

by the accumulation of misfolded proteins and generation of

(A)

(B)

(C)

(D)

Fig 4. MLN2238 inhibits BCL2 and its anti-leukaemic activity is

enhanced by addition of the BH3 mimetic AT-101 (A) B-CLL cells

from 3 patient samples were treated with different concentrations of

MLN2238 for 24 h and the response in BCL2 protein was assessed

by Western Blot. MLN2238 exerted a dose-dependent decrease in

BCL2 as shown in 2 representative patient samples. (B) MLN2238

(12�5 nmol/l) and AT-101 (2�5 lmol/l) caused a decrease in BCL2

protein expression alone and in combination with one another.

(C) Cell death in MLN2238 (M) 12�5 nmol/l treated cells was signifi-

cantly enhanced (P < 0�05) with the addition of AT-101 (A) at

2�5 lmol/l (M+A). This effect is more notable at an AT-101 concen-

tration of 5 lmol/l (M+A1). This was also evidenced by cleavage of

PARP1 on Western blot (D) In all experiments, cells were treated

with either MLN2238, AT-101 or the combination of the two agents

for 24 h. The percentage of tumour cell death was calculated by

using the formula: % apoptosis of control = Tumour cell death –control cell death and reflects the accurate % of cells that underwent

apoptosis in response to the drug(s).

A. Paulus et al


reactive oxygen species upstream of the caspase cascade

(Hideshima et al, 2002; Landowski et al, 2005; Perez-Galan

et al, 2006). Together, these PI-mediated mechanisms con-

tribute to tumour cell death, which is highly contextual and

dependent upon the cell type on which the PI is acting

(Rajkumar et al, 2005).

Our experimental results suggest that the indirect

pro-apoptotic effects of MLN2238 can be augmented when

used in combination with agents designed to disrupt anti-

apoptotic pathways. Such compounds include obatoclax

GX15070, ABT-737, HA14-1 and AT-101, which resemble

the BH3-pro-apoptotic BCL2 molecules and whose binding

activity to BCL2 proper results in tumour cell death

(Konopleva et al, 2008; Tse et al, 2008). The activity of BCL2

inhibitors as single agents in CLL has not made a significant

clinical impact, despite preclinical studies highlighting their

potential for being combined with standard and novel

chemoimmunotherapeutic agents for enhanced tumour kill.

Bortezomib has been investigated in vitro in CLL and other

cancers in combination with several BCL2 inhibitors, includ-

ing obatoclax (Perez-Galan et al, 2008), HA14-1 (Pei et al,

2003) and oblimersen sodium (BCL2 anti-sense oligonucleo-

tide) (O’Connor et al, 2006) with favourable results. How-

ever, these BCL2 inhibitors are primarily selective for BCL2

only, or only weakly bind other anti-apoptotic BCL2 mem-

bers, limiting their use in lymphoid malignancies where

MCL1 and BCL2L1 (BCL-XL) also play a critical role in

tumour cell survival (Beroukhim et al, 2010; Davids & Letai,

2012; Stamelos et al, 2012). Of particular interest is AT-101,

which is the R-(-)-enantiomer of gossypol and is a small

molecule pan-BCL2 inhibitor that binds to the BH3 domains

of anti-apoptotic BCL2 proteins and disrupts their functional

activity. AT-101 displays high binding affinity for BCL2,

BCL2L1 and MCL1 proteins and, in preclinical studies, has

demonstrated impressive cytotoxic activity in a variety of

B-cell malignancies including CLL (James et al, 2006) and

MM (Kline et al, 2008). In our study, AT-101 showed

enhancement of the cytotoxic effects of MLN2238 in vitro.

When combined with AT-101, MLN2238 induced cell death

at a lower dose (12�5 nmol/l) compared to its activity in

single-agent (25–50 nmol/l) form. This observation has clini-

cal implications, signifying that MLN2238’s potential benefit

could be enhanced when rationally combined with agents

targeting complementary oncogenic pathways.

Next, we investigated the in vitro anti-tumour activity of

MLN2238 in combination with the traditional anti-CLL cyto-

toxic agent fludarabine, the widely used glucocorticoid dexa-

methasone and the IMiD lenalidomide. As expected, the

MLN2238 and lenalidomide combination yielded little

improvement in anti-tumour effect over MLN2238 alone

because of the in vitro nature of the experiment and the

requirement of lenalidomide for a host environment to exert

its maximal activity (Chanan-Khan et al, 2011; Masood et al,

2011). However, we observed improved anti-leukaemic activ-

ity of MLN2238 in combination with fludarabine and

moderate effects when combined with dexamethasone.

Fludarabine is a purine nucleoside analog, which is metaboli-

cally converted to its active metabolite, F-ara-ATP (Plunkett

et al, 1993; Ross et al, 1993). In this active form, fludarabine

directly impedes the actions of DNA polymerase and ribonu-

cleotide reductase by competing with dATP, in effect inhibit-

ing DNA synthesis (Parker et al, 1988; Plunkett & Saunders,

1991). It is also capable of integrating itself into the DNA as

a false purine base, resulting in the termination of DNA

synthesis and the activation of the programmed cell death

(A)

(B)

Fig 5. MLN2238 successfully induces tumour cell death when com-

bined with standard anti-CLL agents, such as dexamethasone or flu-

darabine: (A) B-CLL cells from three patient samples were treated

with MLN2238 at 25 nmol/l (M) or at 50 nmol/l (M1), dexametha-

sone (D, 10 lmol/l) or the combination of the two agents for 24 h.

Chronic lymphocytic leukaemia cell death was significant in D+M1

treated cells (P < 0�05) (B) Similarly, cells were treated with

MLN2238 (M or M1), fludarabine (F, 1 lmol/l) or in combination.

Apoptosis was measured by staining for annexin v and propidium

iodide. In both F+M and F+M1 treated cells, we observed a signifi-

cant increase in tumour cell death (P < 0�05) over control cells. Thepercentage of tumour cell death was calculated by using the formula:

% apoptosis of control= Tumour cell death – control cell death and

reflects the accurate% of cells that underwent apoptosis in response

to the drug(s).



response, which it is capable of initiating cellular deconstruc-

tion even in the absence of its incorporation into the DNA

(Spriggs et al, 1986; Huang et al, 1990; Robertson et al,

1993). Fludarabine has been studied in combination with

bortezomib in CLL patient cells in vitro. In this combination,

additive anti-tumour activity was shown to be present

through increased activation of the apoptotic signalling cas-

cade due to upregulation of BAX and downregulation of

inhibitor of apoptosis protein, XIAP (Duechler et al, 2005).

Perhaps these molecular shifts in apoptotic signalling pro-

teins also account for the increased tumour kill that we

observed with the MLN2238-fludarabine combination. These

shifts are being further investigated.

Dexamethasone binds to cytoplasmic glucocorticoid recep-

tors in the target cell, resulting in nuclear translocation of

the receptor and consequent activation of numerous genes

responsible for attenuation of the inflammatory response and

regulation of immune function (Gross et al, 2009). In CLL

cells, the anti-neoplastic effects of dexamethasone have been

shown to be reliant on the expression of the pro-apoptotic

BCL2 family member BCL2L11 and its indirect activation of

BAK1/BAX, thus exposing the anti-CLL activity of the agent

to be partially mediated through the mitochondrial apoptotic

pathway (Iglesias-Serret et al, 2007; Melarangi et al, 2012). In

treating primary patient CLL cells with sub-optimal

(25 nmol/l) concentration of MLN2238 plus dexamethasone,

we observed a marginal increase in apoptosis as compared to

dexamethasone treatment alone. However, when MLN2238

was used at an optimal (50 nmol/l) concentration in combi-

nation with dexamethasone, we noted improved anti-CLL

activity. This highlights the potential for these agents to be

combined and further explored.

Collectively, the data in this report attest to the anti-CLL

activity of MLN2238 in primary CLL cells by its induction of

caspases, PARP1 cleavage, MOMP alteration and inhibition

of BCL2. Further, we have demonstrated that MLN2238 can

be successfully combined in vitro with other anti-leukaemic

agents, resulting in more potent and effective cell death

through alternative/complementary oncogenic pathways.

Thus, these data provide the rationale for additional preclini-

cal experiments and future clinical investigation of MLN2238

in CLL patients.

Acknowledgements

The experiments and analysis carried out in this study were

supported by funding from the Leukemia and Lymphoma

Society (A.C.-K. is a Leukemia and Lymphoma Scholar in

Clinical Research) and the Daniel Foundation of Alabama

(A.C-K). We would also like to thank Kelly Viola for her edi-

torial assistance.

Authors’ contributions

AM designed the research; collected, analysed and interpreted

the data; drafted the article; approved the final draft. AP col-

lected, analysed, and interpreted the data; created the images;

drafted the article; approved the final draft. KCM analysed

and interpreted the data; performed critical revision for

important intellectual content; approved the final draft. ANK

performed experiments, collected, analysed, and interpreted

the data, generated the Figs; and approved the final draft.

DA performed experiments; generated Figs; approved the

final draft. PA analysed and interpreted the data; performed

critical revision for important intellectual content; approved

the final draft. JF analysed and interpreted the data; per-

formed critical revision for important intellectual content;

approved the final draft. CR analysed and interpreted the

data; performed critical revision for important intellectual

content; approved the final draft. GCO analysed and inter-

preted the data; performed critical revision for important

intellectual content; approved the final draft. VR analysed

and interpreted the data; performed critical revision for

important intellectual content; approved the final draft. KS

performed experiments; collected analysed and interpreted

the data and images; drafted the article; approved the final

draft. ACK conceived and designed the research; analysed

and interpreted the data; drafted and performed critical revi-

sion of the article for intellectual content; approved the final

draft.

Funding and disclosures

The authors do not have anything to disclose.

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