pi3kca plays a major role in multiple myeloma and its...

PI3KCA plays a major role in multiple myeloma and itsinhibition with BYL719 decreases proliferation, synergizes withother therapies and overcomes stroma-induced resistance

Feda Azab,1 Shireen Vali,2 Joseph

Abraham,1,3 Nicholas Potter,1,3 Barbara

Muz,1 Pilar de la Puente,1 Mark Fiala,4

Jacob Paasch,4 Zeba Sultana,5 Anuj

Tyagi,5 Taher Abbasi,2 Ravi Vij4 and

Abdel Kareem Azab1

1Department of Radiation Oncology, Cancer

Biology Division, Washington University in Saint

Louis School of Medicine, St. Louis, MO, 2Cell-

works Group Inc., San Jose, CA, 3Saint Louis

College of Pharmacy, St. Louis, 4Section of Stem

Cell Transplant and Leukemia, Division of

Medical Oncology, Washington University School

of Medicine, St. Louis, MO, USA and 5Cellworks

Research India Pvt. Ltd., Bangalore, India

Received 13 August 2013; accepted for

publication 25 November 2013

Correspondence: Abdel Kareem Azab,

Department of Radiation Oncology, Cancer

Biology Division, Washington University in

Saint Louis School of Medicine. 4511 Forest

Park Ave., Room 3103, St. Louis, MO 63108,

USA.

E-mail: [email protected]

Summary

The phosphatidylinositide 3-kinase (PI3K) pathway is activated and corre-

lated with drug resistance in multiple myeloma (MM). In the present study

we investigated the role of PI3KCA (PI3K-a) in the progression and drug

resistance in MM. We showed that the gene expression of PI3KCA isoform

was higher in MM compared to normal subjects. BYL719, a novel and spe-

cific PI3KCA inhibitor inhibited the survival of primary MM cells and cell

lines but not normal peripheral blood mononuclear cells. BYL719 induced

the apoptosis of MM cells and inhibited their cell cycle by causing G1

arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells

cycle signalling, and induced apoptosis signalling in MM cells. Finally,

BYL719 synergized with bortezomib and carfilzomib, and overcame drug

resistance induced by bone marrow stroma. These results were confirmed

using in silico simulation of MM cell lines, BYL719 and bortezomib, and

showed similar trends in survival, proliferation, apoptosis, cell signalling

and synergy with drugs. In conclusion, PI3KCA plays a major role in pro-

liferation and drug resistance of MM cells, the effects of which were inhib-

ited with BYL719. These results provide a preclinical basis for a future

clinical trial of BYL719 in MM as a single agent or in combination with

other drugs.

Keywords: multiple myeloma, PI3KCA (PI3K-a), drug resistance, BYL719,

tumor microenvironment.

Multiple myeloma (MM) is the second most prevalent hae-

matological malignancy with a median survival of 3–5 years

(Kyle & Rajkumar, 2004; Jemal et al, 2005). Despite the

introduction of novel agents, such as bortezomib, only 25%

to 35% of relapsed/refractory MM patients respond to treat-

ment (Jakubowiak, 2012; Mahindra et al, 2012), indicating a

need to improve the therapeutic activity of those agents.

The phosphatidylinositide 3-kinase (PI3K) pathway is dys-

regulated in many tumour types, where it is associated with

a poor prognosis and resistance to multiple therapies; and it

is known to enhance the survival, proliferation and progres-

sion of tumour cells (Chang et al, 2003; Palumbo & Ander-

son, 2011).

The PI3Ks belong to a large family of lipid signalling kin-

ases that are divided into class I, II and III PI3Ks, according

to their molecular structure and cellular regulation. The most

investigated ones in the literature are Class I PI3Ks, which

include: class IA that are activated by growth factor and

cytokine receptors through a tyrosine-kinase-dependent

mechanism [consisting of PI3KCA, PI3KCB (PI3K-b) and

PI3KCD (PI3K-d) isoforms]; and class IB [consisting of

PI3KCG (PI3K-c) only] activated by G-protein coupled-

receptors responding to chemotactic ligands, which control

many cellular functions, such as growth and proliferation,

survival and apoptosis, as well as adhesion and migration

(Rommel et al, 2007). Dysregulation of the PI3K pathway by

loss of PTEN (phosphatase and tensin homolog), the phos-

phatase that degrades phosphatidylinositol-3,4,5-trisphos-

phate (PI-3,4,5-P3), or by activating mutations in the p110-acatalytic subunit of PI3K is one of the most frequent events

in human cancers. However, the role of individual isoforms

of PI3K in development and in signalling is poorly under-

stood (Cantley & Neel, 1999; Luo et al, 2003; Brachmann

et al, 2005).

research paper

ª 2014 John Wiley & Sons LtdBritish Journal of Haematology, 2014, 165, 89–101

First published online 9 January 2014doi:10.1111/bjh.12734

Although no mutations of the PI3K pathway have been

identified in MM patients, the PI3K pathway was shown to be

activated in MM through stimulation with insulin growth fac-

tor 1 (IGF1) and interleukin 6 (IL6) (Hideshima et al, 2001;

Mitsiades et al, 2002), and the activity of the pathway was

shown to increase with the progression of the disease (Hideshi-

ma et al, 2001; Hsu et al, 2001; Mitsiades et al, 2002). It has

been demonstrated that the bone marrow (BM) microenviron-

ment induces drug resistance in MM cells and other haemato-

logical malignancies, through activation of the PI3K/AKT

pathway (Zhang et al, 2003; Azab et al, 2009a; Podar et al,

2009; Weisberg et al, 2012). Therefore, the PI3K pathway has

been identified as a desirable target to overcome drug resistance

in MM, and inhibitors of different kinases in the PI3K pathway

have been suggested for treatment of MM in combination with

other drugs, including the AKT inhibitor perifosine (Hideshi-

ma et al, 2006), the PKC–AKT inhibitor enzastaurin (Neri

et al, 2008), the mammalian target of rapamycin (mTOR)-

PI3K inhibitor NVP-BEZ235 (McMillin et al, 2009) and the

pan-PI3K inhibitor NVP-BKM120 (Zheng et al, 2012).

The PI3K pathway regulates cellular functions relevant to

tumourogenesis, but unfortunately, clinical studies of broad

PI3K inhibitors have been plagued by toxicity (Zheng et al,

2011). More specific approaches to inhibit specific isoforms

in MM were performed; PI3KCD was inhibited using the

specific inhibitor CAL-101, which overcame MM cell growth

conferred by IL6, IGF1, and bone marrow stromal cell cocul-

ture (Ikeda et al, 2010). In chronic lymphocytic leukaemia

(CLL) patients, CAL-101 showed acceptable toxicity, positive

pharmacodynamic effects, and favourable clinical activity in

heavily pretreated patients, including patients with refractory

disease, bulky lymphadenopathy and poor-prognosis cytoge-

netics (Furman et al, 2010).

PI3KCA has attracted considerable interest as a drug target

since its identification as an oncogene and as the most fre-

quently mutated oncogene in breast and endometrial cancers

(Samuels et al, 2004; Denley et al, 2008). In the present

study we investigated the role of PI3KCA in disease progres-

sion and drug resistance in MM, by inhibiting it with a novel

specific PI3KCA inhibitor (BYL719), and tested its effect on

survival, apoptosis, cell cycle, sensitivity to drugs and interac-

tion with BM microenvironment of MM cells.

Methods

Reagents

BYL719 was purchased from SelleckChem (Houston, TX,

USA), monoclonal antibodies (mAbs) for Western blotting

from Cell Signaling Technologies (Danvers, MA, USA), pro-

pidium iodide (PI), RNAase and 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide (MTT) solution from

Sigma-Aldrich (Saint Louis, MO, USA), Calcein-AM from

Invitrogen (Carlsbad, CA, USA), Annexin-V/PI assay from BD

Biosciences (San Jose, CA, USA).

Cells

The MM cell lines (OPM1, OPM2, RPMI8226, U266, MM1s,

MM1R and NCI-H929) and BM stromal cell (BMSC) line

HS5 were a kind gift from Dr. Irene Ghobrial, Dana-Farber

Cancer Institute, Harvard Medical School, Boston, MA. All

cells were cultures at 37°C, 5% CO2, in RPMI 1460 media

supplemented with 10% fetal bovine serum (FBS), penicillin

and streptomycin, except HS5 which was cultured in Dul-

becco’s modified Eagle medium supplemented with 20%

FBS.

Primary CD138+ cells were isolated from bone marrow

aspirates of MM patients, and peripheral blood mononuclear

cells (PBMCs) were isolated with pheresis leukopaks from

the Siteman Cancer Center, Washington University in Saint

Louis, as previously described (Azab et al, 2012a,b).

Informed consent was obtained from all patients, with

approval from the Washington University Medical School

Institutional Review Board committee and in accord with the

Declaration of Helsinki.

Gene expression analysis

To determine the gene expression of the different isoforms of

PI3K in MM, smouldering myeloma, monoclonal gammopa-

thy of undetermined significance (MGUS), normal subjects

and MM cell lines, we used published datasets GSE2658 and

GSE5900 (Zhan et al, 2006; Keats et al, 2007). To determine

PI3KCA, PI3KCB, PI3KCD and PI3KCG gene expression we

used the probes 204369_at, 212688_at, 203879_at and

206369_s_at, respectively.

Cell viability assay

MM cell lines (OPM1, OPM2, RPMI8226, U266, MM1s,

MM1R and NCI-H929), primary cells and PBMCs were cul-

tured with BYL719 (0–2�5 lmol/l) alone or in combination

with bortezomib (0–5 nmol/l), or carfilzomib (0–2�5 nmol/l)

for 48 h. The range of concentrations of the proteasome

inhibitors and BYL719 were chosen to be around (lower and

higher than) the 50% inhibitory concentration (IC50) of

each drug alone. In some cases, MM1s cells were co-cultured

with a previously prepared monolayer of BM stroma and

treated with BYL719, with or without bortezomib or carfilzo-

mib. Cell proliferation was assessed by MTT assay as previ-

ously described (Azab et al, 2012a). Briefly, the MTT

solution was added to the cells at 44 h from the start of

treatment, and after 4 h the stop solution was added and the

absorption in wells was read at 570 nm.

Synergism calculation

To calculate the synergism with other drugs we used the Bliss

independence model, which is defined by the equation:

Exy = Ex + Ey � ExEy, where Exy is the additive effect of

F. Azab et al

90 ª 2014 John Wiley & Sons LtdBritish Journal of Haematology, 2014, 165, 89–101

drugs x and y as predicted by their individual effects, Ex and

Ey. In this case, Exy would be the effect (fractional survival)

of the bortezomib or carfilzomib in combination with

BYL719, and Ex and Ey the fractional survival of cells

exposed to the bortezomib or carfilzomib alone, respectively.

The effect is synergistic when Exy > 0.

Apoptosis assay

MM1s cells (0�5 9 106 cells/ml) were cultured with increased

concentrations of BYL719 (0–2�5 lmol/l) for 48 h. Cells were

then stained with Annexin/PI for 15 min, washed with PBS,

and analysed by flow cytometry, as previously described

(Azab et al, 2012a). Briefly, cells were suspended in Annexin-

binding buffer, incubated with Annexin-V-FITC for 30 min

on ice, stained with PI for 10 min and analysed by flow

cytometry.

Cell cycle analysis

MM1s (0�5 9 106 cell/ml) were cultured with increased con-

centrations of BYL719 (0–2�5 lmol/l) for 24 h as previously

described (Azab et al, 2012b). Briefly, cells were then fixed

with 70% ethanol, washed, RNA was degraded by RNAase,

the DNA was stained with 5 lg/ml PI (Sigma, St. Louis,

MO), and cells were analysed by flow cytometry, as previ-

ously described (Azab et al, 2012a). Briefly, cells were fixed

and permeabilized with ice cold ethanol and washed; the

RNA was degraded with RNase, and the DNA was stained

with PI for 10 min, and analysed by flow cytometry.

Western blotting

To test the effect of BYL719 on the PI3K and adhesion sig-

nalling, MM1s cells were treated with BYL719 (0–2�5 lmol/l)

for 6 h, in some cases MM1s and NCI-H929 cells were trea-

ted with BYL719 0�5 lmol/l with or without bortezomib

5 nmol/l and Carfilzomib 5 nmol/l for 6 h. Cells were then

washed and lysed, and whole-cell lysates were subjected to

sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE) as described previously (Azab et al, 2012b).

Briefly, membranes were blotted overnight at 4°C with mAb

for pAKT, pS6R, pGSK, pFAK, pSRC, pCofilin and devel-

oped using a chemiluminescence assayTubulin was used as

a loading control.

To test the effect of BYL719 on apoptosis and cell cycle

signalling, MM1s and NCI-H929 were treated with BYL719

(0–2�5 lmol/l) for 24 h, in some cases BYL719 0�5 lmol/l

was combined with bortezomib 5 nmol/l or carfilzomib

5 nmol/l. Cells were then lysed and whole-cell lysates were

subjected to SDS-PAGE, then the membranes were blotted

overnight at 4°C, with mAb for cleaved-Caspase-3, cleaved-

Caspase-9, cleaved-PARP, p-JNK, pCyclin E1, pRb, P27, and

a-Tubulin. Proteins were detected using chemiluminescence

(Azab et al, 2012a).

Adhesion of MM cells to BMSCs

For the adhesion assay, plates were coated with a confluent

monolayer BMSCs generated by plating 1 9 104 cells/well in

96-well plates overnight. The next day MM1s cells

(1 9 106 cells/ml) were serum starved for 6 h, pre-labelled

with calcein-AM, treated with increasing concentrations (0,

0�5 and 1 lmol/l) of BYL719, then added to 96-well plates

pre-coated with a monolayer of BMSCs. Cells were co-cul-

tured for 1 h at 37°C and non-adherent cells were washed.

Adherent cells were detected by measuring the fluorescence

intensity in the wells using a fluorometer (Ex / Em = 485/

520 nm) as previously described (Azab et al, 2011, 2012b).

In silico simulation of biological effect

Cancer simulation model description. The predictive compu-

tational studies of BYL719 and bortezomib were performed

using the functional cancer physiology aligned simulation

model of plasma cells from Cellworks Group Inc. This kinet-

ics-driven simulation model is a comprehensive representa-

tion of signalling and metabolic pathways and integrates all

cancer phenotypes, such as proliferation, apoptosis, viability,

angiogenesis, tumour metabolism and metastasis. The simu-

lation ability allows ‘what-if’ studies and functional screening

of drugs with complete transparency into the underlying net-

work of pathways at the bio-marker level. The simulation

model has been extensively validated through prospective

and retrospective studies showing good correlation between

predictive readouts and wet-lab assays (Cirstea et al, 2010;

Roy et al, 2010; Kannaiyan et al, 2011; Rajendran et al, 2011;

Shanmugam et al, 2011).

The simulation model has been developed through a bot-

tom-up approach by manual inference of bio-chemical sig-

nalling networks from research and aggregation using

mathematical representation. The manual inference and rep-

resentation of functional relationships enables handling of

contradictory datasets and connecting dots across research

studies. The simulation model is constantly enhanced and

the current version represents over 6200 species with cros-

stalk interactions exceeding multiples of number of species.

The model is a comprehensive coverage of the kinome, tran-

scriptome, proteome and, to some extent, metabolomic com-

ponents. Selected examples of coverage include signalling

pathways, such as growth factors like EGFR, PDGFRA,

FGFR, MST1R (c-MET), VEGFR and IGF1R, cell cycle regu-

lators, mTOR signalling, TP53 signalling cascade, HIF signal-

ling, apoptotic machinery, DNA damage repair, ER-stress,

autophagy, Ubiquitin proteasome machinery, cytokine path-

ways, such as IL1, IL4, IL6, IL12 and TNF, lipid mediators

and tumour metabolism and others. The modelling of the

time-dependent changes in the fluxes of the constituent

pathway was performed utilizing modified ordinary

differential equation (ODE) and mass action kinetics of

proliferation (CDK4-CCND1, CDK2-CCNA, CDK2-CCNE,

PI3KCA Inhibition Sensitizes Myeloma Cells to Therapy

ª 2014 John Wiley & Sons Ltd 91British Journal of Haematology, 2014, 165, 89–101

CDC2-CCNB1), viability (survival markers/apoptosis mark-

ers) and apoptosis [BAX, CASP3, CASP8, PMAIP1 (NOXA)

and BCL2L11 (BIM)].

Creation of cell line models. To create simulation model

characterized equivalents of cell lines, the mutation informa-

tion was derived from resources such as Sanger and other lit-

erature research and functionally introduced (Finelli et al,

1999; Ikediobi et al, 2006; Cassinelli et al, 2009; Steinbrunn

et al, 2011). The created simulation cell lines models were

validated against a set of experimental studies to confirm the

definition accuracy (see Table I).

Simulation of BYL719 and bortezomib individually and in

combination. The drug was introduced in the simulation

model after deriving the mechanism of action (MOA) of

each drug based on published research (Adams et al, 1999;

Piperdi et al, 2011; Furet et al, 2013; Young et al, 2013) and

validation of the mechanism across retrospective studies. The

directly inhibited/activated primary (and secondary as well as

tertiary in some cases) targets of the compound reported in

the experimental literature were modulated with experimen-

tally-determined kinetic constants. In this study, BYL719 has

been represented as a PI3KCA inhibitor and bortezomib as a

proteasome inhibitor. The drug concentration is explicitly

assumed to be post-ADME (Absorption, Distribution,

Metabolism and Excretion).

Simulation protocol. The cancer simulation model was simu-

lated and initialized to a normal physiological control state

wherein all biological species attain steady state. This non-

transformed plasma cell was triggered to represent corre-

sponding cell lines by overlaying mutation information on

the network that introduces mutations on different oncoge-

nes and/or tumour suppressors and other genetic and

epigenetic changes that modulate the functional levels of

genes and proteins. Finally, the drug agents were added

individually and in combination by introducing the primary

biological mechanisms. The simulation concentration ‘C’ for

each drug is the IC30 value with respect to viability. Post

this, the study is simulated and endpoint markers and

phenotypes assayed.

Results

PI3KCA isoform plays a major role in MM Progressionand BYL719 inhibits MM proliferation

We analysed the gene expression of PI3KCA (ID

204369_at), PI3KCB (ID 212688_at), PI3KCG (ID

206369_s_at) and PI3KCD (ID 203879_at) in MM patients

based on published datasets from the Gene Expression

Omnibus by Zhan et al (2006), to test which one of the

four isoforms is the most dominant one in MM

patients. We found that the gene expression of PI3KCA and

PI3KCB isoforms was significantly higher than PI3KCG

and PI3KCD (Fig 1A). Therefore, we analysed PI3KCA and

PI3KCB expression of MM disease with the progression of

the disease (MGUS, smouldering myeloma and MM) com-

pared to normal subjects, and found that both genes were

upregulated; however, the fold of increase of expression of

PI3KCA was greater than PI3KCB in smouldering myeloma

and MM, as compared to normal subjects (Fig 1B). This

indicates that the PI3KCA isoform plays a more significant

role in the progression of MM. To investigate the role of

PI3KCA isoform in MM, we used the novel PI3KCA selec-

tive inhibitor BYL719 whose chemical structure and speci-

ficity of inhibition of PI3KCA isoform compared to the

other isoforms have been previously described (Furet et al,

2013). We examined the effect of BYL719 on the prolifera-

tion of primary MM cells isolated from three MM patients,

and found that BYL719 inhibited their proliferation at an

IC50 of approximately 1 lmol/l (Fig 1C); in contrast,

BYL719 did not affect the proliferation of PBMCs from

three healthy donors (Fig 1D). Furthermore, we confirmed

the effect of BYL719 in MM cell lines and concurrently in

the equivalent simulation models. By treating OPM1,

OPM2, RPMI, U266, MM1s, MM1R and H929 cells with

BYL719 (0–2�5 lmol/l), BYL719 inhibited the proliferation

of all MM cell lines tested in a different manner (Fig 1E).

We analysed the correlation between the activity of PI3KCA

predicted in the in silico simulation technology for cell lines

with different levels of sensitivity towards BYL719, based on

the in vitro proliferation assay (OPM2-highly resistant,

RPMI-moderately resistant, MM1s-moderately sensitive,

H929-highly sensitive) and the percentage of dead cells fol-

lowing treatment with 1 lmol/l BYL719. This concentration

was chosen, as it was the IC50 of all of the three patient

samples used. We found that the in silico-predicted PI3KCA

activity in the cell lines was exponentially correlated with

the killing induced by the PI3K inhibitor BYL719 in vitro

(Fig 1F).

Table I. The mutation components for the cell lines used.

Cell line Tissue type Definition

MM1S B lymphoblast,

MM

KRAS, EGFR, CDKN2A, CDKN2B,

CDKN2C, MYC, MAF, NR3C1,

WHSC1, TRAF3, IRF8, NOX3,

PRR5, SIPR1, VCAM1, WWOX

RPMI8226 B-lymphocyte,

MM

KRAS, Tp53, CDKN2A, CDKN2C,

SOCS1, TRAF3, WWOX, MAF,

MALT1, BCL2, PTPN6

NCI-H929 B-lymphocyte,

MM

KRAS, CDKN2A, MYC, MUC1,

MCL1, BCL9

OPM2 B-lymphocyte,

MM

KRAS, TP53, PTEN, SOCS3, SOCS1,

CDH1, CDKN2A, CDKN2C,

RASSF1A, RARB, WHSC1, FGFR3,

MALT1,

MYC, BCL2

F. Azab et al


BYL719 inhibits PI3K pathway and induces cell cyclearrest in MM cells

Mechanistically, we tested the effect of BYL719 on the PI3K

signalling pathway in both MM1s and NCI-H929 cell lines

and equivalent simulation models. We found that it signifi-

cantly decreased the activation of the PI3K signalling proteins

(pAKT, pS6R, and pGSK) (Fig 2A), the same pattern was

predicted in silico, in which BYL719 decreased the expression

of pAKT, pS6R, and pGSK in a dose-dependent manner

(Fig 2B). We further examined the role of the PI3KCA iso-

form in the proliferation and cell cycle MM1s cells, it was

found that BYL719 induced G1 arrest in which the G1-phase

was increased and the S-phase was decreased following treat-

ment with BYL719 in a dose-dependent manner (Fig 2C).

This correlated with the simulation predictions; where

BYL719 (0–2�5 lmol/l) inhibited MM proliferation of MM1s

cells in a dose-dependent manner (Fig 2D). We confirmed

these findings by testing the effect of BYL719 on the down-

stream signalling of cell cycle proteins involved in the transi-

tion from G1 to S phase in MM1s and NCI-H929 and found

that it decreased the levels of pCyclin-E1 and pRb; and

increased of the levels the cell cycle inhibitory protein P27 in

a dose dependent manner (Fig 2E). Similarly, in silico studies

confirmed the effect of BYL719 on pCyclin E1 and P27, and

it further predicted the inhibition of other cell-cycle proteins

including CDK4-Cyclin D complex, Myc-Max complex and

CDK1-Cyclin B complex by BYL719 in a dose-dependent

manner (Fig 2F).

BYL719 induces Apoptosis in MM cells

We tested the effect of BYL719 on apoptosis of MM1s cells

using simulation and cell line studies. It was found that

BYL719 (0–2�5 lmol/l) increased the fraction of apoptotic

(Annexin+/PI+) and early apoptotic (Annexin+/PI�) MM1s

cells in a dose-dependent manner, as detected by Annexin/PI

staining and analysed by flow cytometry (Fig 3A). Similar

results were predicted in silico, in which BYL719 induced

apoptosis in MM1s cells in a dose-dependent manner

(Fig 3B).

To investigate the cellular mechanism of apoptosis

induced by BYL719 we investigated its effect on apoptosis-

related proteins. It was found that BYL719 induced the

cleavage of Caspase-3, Caspase-9 and PARP, in a dose-

dependent manner (Fig 3C). This correlated again with the

in silico simulation predictions, in which BYL719 induced a

(A) (B)

(C) (D)

(E) (F)

Fig 1. PI3KCA isoform plays a major role in

MM Progression and BYL719 inhibits MM

proliferation. (A) Gene expression of PI3KCA

(ID 204369_at), PI3KCB (ID 212688_at),

PI3KCG (ID 206369_s_at) and PI3KCD (ID

203879_at) in MM patients based on published

datasets from the Gene Expression Omnibus

(Zhan et al, 2006). (B) Gene expression of

PI3KCA (ID 204369_at) and PI3KCB (ID

212688_at) at different stages of MM progres-

sion (MGUS, Smouldering myeloma and MM)

normalized to the expression in healthy sub-

jects, based on published datasets from the

Gene Expression Omnibus (Zhan et al, 2006).

The effect of increasing concentrations of

BYL719 (0–2�5 lmol/l) on the proliferation of

(C) primary MM cells isolated from the BM of

three MM patients, (D) three healthy PBMC,

and (E) MM cell lines (OPM1, OPM2, RPMI,

U266, MM1s, MM1R and H929) by MTT

assay for 48 h. (F) The correlation between the

predicted activity of PI3KCA by the in silico

technology of the chosen cell lines (OPM2,

RPMI, MM1s, H929) and the dead cells at

1 lmol/l of BYL719 treated for 48 h. MM,

multiple myeloma; MGUS, monoclonal gamm-

opathy of undetermined significance; PBMC,

peripheral blood monoclonal cells; MTT, 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-

um bromide.



(A) (B)

(C) (D)

(E)

(F)

Fig 2. BYL719 inhibits the PI3K pathway and induces cell cycle arrest in MM cells. (A) The effect of treatment for 6 h with increasing concentra-

tions of BYL719 (0–2�5 lmol/l) on the PI3K signalling- p-Akt, p-S6R, p-GSK in MM1s and NCI-H929 cells by Western blotting. (B) the expres-

sion of the PI3K signalling; p-AKT, p-GSK and p-S6R by the in silico system after applying increasing concentration of BYL719 (0–2�5 lmol/l).

(C) The effect of increasing concentrations of BYL719 (0–2�5 lmol/l) for 24 h on the cell cycle of MM1s cells. (D) The effect of BYL719 on the

proliferation of MM1s cells in silico after applying increasing concentration of BYL719 (0–2�5 lmol/l). (E) The effect of BYL719 on cell cycle sig-

nalling; p-Cyclin E, p-RB, P27 by Western blotting in MM1s and NCI-H929 cells. (F) The effect of BYL719 (0–2�5 lmol/l) on cell cycle proteins

and complexes; p-CyclinE, p27 CDK4-CyclinD complex, Myc-Max Copmlex, CDK1-Cyclin B complex in silico.

F. Azab et al


dose-dependent expression of the cleaved form of Caspase-3,

Caspase-9 and PARP (Fig 3D).

BYL719 synergizes with proteasome inhibitors

We tested the effect of the combination of BYL719 (0, 0�5,0�75 and 1 lmol/l) with bortezomib (0, 3 and 5 nmol/l) on

the proliferation of MM1s cells. It was found that the combi-

nation of the two drugs decreased the surviving fraction of

MM1s cells more than each of the drugs alone (Fig 4A), the

Bliss synergy index was positive for all combination points

except the (BYL719 1 lmol/l, bortezomib 5 nmol/l), in

which the killing effect of each drug alone was profound,

which made it difficult to achieve synergy. Similar results

were predicted in silico, in which the combination of BYL719

(0, 0�5, 0�75, 1 lmol/l) and bortezomib (3, 5 nmol/l)

inhibited the proliferation of MM1s cells (Fig 4B). The in sil-

ico studies predicted that the drugs synergize on the apopto-

tic markers. Mechanistically, we confirmed these results by

testing the effect of the combination of both drugs on the

PI3K signalling pathway and found that, while bortezomib

increased pAKT and pS6R in MM1s and NCI-H929 cells,

BYL719 abolished the bortezomib-induced increase of pAKT

and pS6R. Moreover, we tested the effect of the combination

on the apoptosis signalling and found that the combination

of the drugs increased cleavage of PARP, caspase-3 and cas-

pase-9 more than each of the drugs alone (Fig 4C). Although

the bortezomib-induced activation of p-AKT and p-S6R was

not predicted in silico, similar findings were predicted in

apoptosis signalling, in which the combination of the drugs

induced caspase-3, caspase-9 and PARP cleavage more than

each of the drugs alone (Fig 4D).

(A) (B)

(C) (D)

Fig 3. BYL719 induces Apoptosis in MM cells. (A) The effect of increasing concentrations of BYL719 (0–2�5 lmol/l) for 48 h on the apoptosis

of MM1s cells analysed by Annexin/PI staining. (B) The effect of increasing concentrations of BYL719 (0–2�5 lmol/l) on the apoptosis of MM1s

cells in silico. (C) The effect of BYL719 on the apoptosis signalling; cleaved PARP, caspase 3, caspase 9 by Western blotting. (D) Expression level

of apoptosis signalling; cleaved Caspase3, Caspase 9 and cleaved PARP in silico. MM, multple myeloma; PI, propidium iodide.



(A) (B)

(C) (D)

(E) (F)

Figure 4. BYL719 synergizes with Proteasome inhibitors in inhibiting theproliferation of MM cells. (A)The effect of the combination of increasing

concentrations of BYL719 (0, 0�5, 0�75 and 1 lmol/l) with increasing concentrations of bortezomib (0, 3 and 5 nmol/l) on the proliferation of

MM1s cells by MTT. (Insert: Bliss synergy calculation results). (B) The effect of the combination of BYL719 (0, 0�5, 0�75, 1 lmol/l) and bortezo-

mib (0, 3 and 5 nmol/l) on the proliferation of MM1s cells in silico. (C) The effect of the combination of BYL719 0�5 lmol/l with bortezomib

5 nmol/l on the downstream signalling of PI3K (pAKT and pS6R) and apoptosis (cleaved PARP, caspase-3 and caspase-9) in in MM1s and NCI-

H929 cells by Western blotting. (D) The effect of the combination of BYL719 and bortezomib on the expression level of PI3K (pAKT and pS6R)

and apoptosis (cleaved PARP, caspase-3 and caspase-9) in MM1s cells in silico. (E) The effect of the combination of increasing concentrations of

BYL719 (0, 0�5, 0�75 and 1 lmol/l) with increasing concentrations of carfilzomib (0, 1�25 and 2�5 nmol/l) on the proliferation of MM1s cells by

MTT. (Insert: Bliss synergy calculation results) (F) The effect of the combination of BYL719 0�5 lmol/l with carfilzomib 5 nmol/l on apoptosis

signalling (pJNK, cleaved PARP, cleaved caspase-3 and cleaved caspase-9) in MM1s cells by Western blotting. MM, multiple myeloma; Bort, bort-

ezomib; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

F. Azab et al


We further tested the effect of the combination of BYL719

(0, 0�75 and 1 lmol/l) and carfilzomib (0, 1�25 and

2�5 nmol/l). It was found that, similar to Bortezomib, the

combination of the drugs decreased the surviving fraction of

MM cells more than each of the drugs alone (Fig 4E), and

the Bliss synergy index was positive for all combination

points. BYL719 enhanced the activation of pJNK induced by

carfilzomib, and the combination of the drugs induced more

cleavage of PARP, Caspase-3 and Caspase-9 compared to

each alone (Fig 4F).

BYL719 inhibits adhesion to BM stroma and overcomesdrug resistance to proteasome inhibitors induced bystroma

We tested the effect of BYL719 (0, 0�5, 1 lmol/l) on the

interaction of MM1s cells with BMSCs, and it was found that

BYL719 decreased the adhesion of MM1s cells to BMSCs in

a dose-dependent manner (Fig 5A). We confirmed these

results by testing the effect of BYL719 on the adhesion sig-

nalling and found that it decreased the activation of adhesion

signalling, such as pFAK, pSRC and pCofilin in a dose-

dependent manner (Fig 5B). These findings were partially

predicted in silico, in which BYL719 decreased the expression

of pFAK, pSRC and pCofilin, but only pCofilin showed inhi-

bition in a dose-dependent manner (Fig 5C). The in silico

studies further predicted the inhibition of the small GTPases

Rho, Rac and Cdc42 (Fig 5D), in which Rho and CDC42,

but not Rac, showed inhibition in a dose-dependent manner.

To test the effect of BYL719 on drug resistance induced

by the BM stroma, MM1s cells were co-cultured with

BMSCs, treated with BYL719 (0 and 0�75 lmol/l) in combi-

nation with bortezomib (0 and 3 nmol/l) or carfilzomib (0

and 2�5 nmol/l) for 48 h. It was found that co-culture with

BMSCs increased the surviving fraction of MM cells after

treatment with bortezomib (Fig 5E) and carfilzomib (Fig 5F),

as a drug resistance mechanism. The combination of the two

drugs with BYL719 overcame the resistance induced by the

stroma and reduced the surviving fraction to the values

observed for treatment without the presence of stroma

(Figs 5E and F).

Discussion

Recent advances in the treatment of MM with novel thera-

peutic agents, such as thalidomide, bortezomib and lenalido-

mide, have significantly enhanced the response of MM

patients to therapy (Ghobrial et al, 2007). In treating patients

with relapsed or refractory myeloma, a combination of dif-

ferent agents can achieve a better response even when the

disease is resistant to therapy (Palumbo & Anderson, 2011).

However, many patients do not respond and/or acquire drug

resistance to these agents, emphasizing the need for novel

strategies to improve the treatment. In the present study, we

suggest a novel strategy to improve the treatment of MM

and overcome the drug resistance for the current therapeutic

agents by specific inhibition of PI3KCA in MM cells.

Clinical studies of pan-PI3K inhibitors have been plagued

by toxicity, such as inhibiting microtubule dynamics upon

direct binding to tubulin (Brachmann et al, 2012); therefore,

and to minimize the side effects, we focused on inhibition of

a single PI3K isoform. The gene expression of the different

PI3K isoforms in MM patients were investigation, and it was

found that the alpha and beta isoforms showed higher

expression than the gamma and delta isoforms. However, the

fold-change of expression of the alpha isoform in MM

patients was higher than the beta isoform. Therefore, we

focused on PI3KCA as the more dominant PI3K isoform in

MM, and used the small molecule inhibitor BYL719 as a

selective PI3KCA inhibitor (Furet et al, 2013).

BYL719 inhibited the proliferation of MM Patients with

an IC50 of about 1 lmol/l in the three patients, while none

of three normal PBMC controls reached an IC50, and actu-

ally no effect was observed on the proliferation of PBMCs in

this dose range. This indicated that the PI3KCA can serve as

a selective therapeutic target in MM, due to the large thera-

peutic window demonstrated in these studies, unlike other

pan-PI3K inhibitors(Bendell et al, 2012). We tested the effect

of BYL719 on the proliferation of several MM cells lines, and

found that the effect can vary, from very high sensitivity

(such as H929) to significant resistance (such as OPM2).

Using the simulation-based predictive studies together with

the cell line studies; we modelled and simulated several MM

cell lines according to their published features including

mutation and activity of signalling pathways. The in silico

predictions were blindly validated by the cell line studies,

which included correlation across H929 (highly sensitive),

MM1S (moderately sensitive), RPMI (moderately resistant)

and OPM2 (highly resistant) cells. The dominance of each

PI3K isoform was calculated in each of these cell lines. We

found that the higher the PI3KCA activity in the cell lines,

the more significant is the effect of BYL719 on the prolifera-

tion of the cells. The PI3KCA activity (predicted in silico)

was exponentially correlated with the inhibitory effect of the

proliferation of MM cells by BYL719 (found in vitro),

R2 = 0�9833. This indicates the selectivity of the inhibitor to

PI3KCA, as previously described (Furet et al, 2013). More-

over, we found that BYL719 inhibited the PI3K signalling,

decreased the proliferation and cell cycle, and induced apop-

tosis in MM cells as a single agent. Similar results were pre-

dicted by the in silico system, indicating that that the in silico

system provides an accurate prediction of the activity of the

drug in MM and a potential engine to design novel thera-

peutics.

Bortezomib brought a significant improvement in the

treatment of MM; however, studies showed that 60% of

patients will develop resistance to bortezomib (San Miguel

et al, 2008). Preclinical models suggested that resistance to

bortezomib was shown to occur through activation of the

PI3K pathway and increased activity of pAKT in MM cells,



which is attenuated in the bone marrow microenvironment

(Hideshima et al, 2001; Azab et al, 2009a). In the current

study, we suggested combining BYL719 with bortezomib to

overcome resistance to bortezomib in MM. In vitro and in

silico tests showed that BYL719 synergized with bortezomib

and inhibited proliferation of MM cells through inhibition of

(A) (B)

(C) (D)

(E) (F)

Fig 5. BYL719 inhibits adhesion to BM stroma and overcomes drug resistance induced by stroma. (A) The effect of increasing concentrations of

BYL719 (0, 0�5 and 1 lmol/l) on the adhesion of MM cells to plates coated with BMSCs. (B) The effect of BYL719 (0, 0�5 and 1 lmol/l) on

adhesion signalling (P-FAK, p-SRC and p-Cofilin) in MM cells by western blotting. The effect of increasing concentration of BYL719 (0–2�5 lmol/l) on (C) adhesion signalling (p-FAK, p-SRC and p-Cofilin) and (D) Rho GTPases (Rho, Rac and Cdc42) in MM cells, in silico. The

effect of the combination of BYL719 with (E) bortezomib or (F) carfilzomib, on the proliferation of MM cells in co-culture with BMSCs, analysed

by MTT assay (inserts in E and F are the numerical values of the experimental data). MM, mutliple myeloma; BM, bone marrow; BMSC, bone

marrow stomal cells; BYL, BYL719; Bort, bortezomib; Carf, carfilzomib; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

F. Azab et al


the PI3K activation induced by Bortezomib. Moreover, the

combination of the drugs induced apoptosis and cleavage of

caspase-3, caspase-9 and PARP more than each of the drugs

alone.

Carfilzomib is an emerging therapy which was recently

approved for the treatment of MM (Kortuem & Stewart,

2013). Previous studies showed that some bortezomib-resis-

tant cell lines (Kuhn et al, 2009) and patients (Vij et al,

2012) were responsive to carfilzomib. Carfilzomib induced

apoptosis by activation of pJNK (Dasmahapatra et al, 2012);

in this study we tested the effect of the combination of

BYL719 with carfilzomib on the proliferation and apoptosis

of MM cells, and found that the drugs synergize through

activation of p-JNK and induction of more caspase and

PARP cleavage in MM cells.

The interaction with the BM microenvironment was

shown to play a crucial role in the progression of drug resis-

tance in MM cells (Azab et al, 2009a, 2012a) and other hae-

matological malignancies (Azab et al, 2013). Adhesion of

MM cells to stromal cells was shown to activate adhesion sig-

nalling and the PI3K pathway and induce proliferation and

drug resistance (Nefedova et al, 2003; Azab et al, 2009a,b).

In this study we have shown that BYL719 inhibited the adhe-

sion of MM cells through inhibiting the activation of adhe-

sion-related proteins; p-FAK, p-SRC and p-Cofilin, and

similar results were predicted in silico. In addition, the in sil-

ico studies predicted that the effect was also facilitated

through the inhibition of the small GTPases (mainly Rho

and CDC42, but not Rac). These results are in agreement

with our previous findings that Rho plays a more important

role than Rac in MM cell interaction with the BM microen-

vironment(Azab et al, 2009b).

Previous studies showed that MM drug resistance to

bortezomib occurs through activation of the PI3K pathway,

and increased when MM cells are cultured with stroma

(Azab et al, 2009a). The microenvironment-induced drug

resistance to bortezomib was e reversed when inhibiting the

direct interaction of MM cells with the BM microenviron-

ment (Azab et al, 2009a, 2012a); therefore, we tested the

effect of inhibition of PI3KCA on the stroma-induced resis-

tance to bortezomib in MM cells. As expected, the co-cul-

ture of MM cells with BMSCs induced resistance to

bortezomib, and BYL719 overcame the resistance induced

by the stroma and reduced the surviving fraction to the

values similar to the ones observed for treatment without

presence of stroma.

Previous studies have shown that BM stroma induced

resistance to carfilzomib in Waldenstrom Macroglobulinae-

mia (Sacco et al, 2011) and in CLL (Gupta et al, 2013).

Therefore, we tested the effect of BM stroma on the sensitiv-

ity of MM cells to carfilzomib, and here we first report that,

similar to other haematological malignancies, BM stroma

induced drug resistance to carfilzomib. However, BYL719

abolished the stroma-induced resistance to carfilzomib and,

when the two drugs were combined in the presence of

stroma, the surviving fraction was reduced to the values

lower than the ones observed for each treatment without

presence of stroma.

In summary, this study showed that the PI3KCA isoform

plays a significant role in the progression and drug resistance

in MM, and that its inhibition with BYL719 reduces prolifer-

ation, inhibits cell cycling and induces apoptosis in MM

cells. Moreover, it showed that BYL719 synergizes with bort-

ezomib and carfilzomib, and overcomes the drug resistance

induced by BM stroma. In addition, we have developed an

in silico system that simulates MM cells and can accurately

predict the effect of drugs on function and signalling in MM.

These results provide a preclinical basis of future clinical tri-

als of BYL719 in MM as a single agent or in combination

with other drugs.

Author contributions

FA: Performed research, analysed data, designed research and

wrote the paper. BM, PDLP, JA, NP, MF, JP, SV, ZS, AT,

TA and RV: Performed research and analysed data. AKA:

Performed research, analysed data, designed research, wrote

the paper and supervised the study.

Conflict of interest

SV and TA work for Cellworks Group, Inc., 2025 Gateway

Place, Suite 265, San Jose, CA 95110, USA,which is develop-

ing the predictive simulation models for rationally designing

therapeutics. ZS and AT work for Cellworks Research India

Limited, Whitefield, Bangalore 560 066, India, which is a

fully owned subsidiary of Cellworks Group Inc. All the other

authors have no conflict of interest.

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