Lenalidomide synergizes with dexamethasone to induce growth arrest and apoptosis of mantle cell lymphoma cells in vitro and in vivo Zhengzi Qian*1, 2,

Lenalidomide synergizes with dexamethasone to induce growth arrest and apoptosis of mantle cell lymphoma cells in vi

tro and in vivo Zhengzi Qian*1, 2, Zhen Cai 2, Luhong Sun 2, Huaqing Wang 1, Qing Yi 2, Michael Wang 2, and Liang Zhang 21 Department of Lymphoma, Tianjin Medical University Cancer Hospital and Institute, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China 2 Department of Lymphoma and Myeloma, Division of Cancer Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

BackgroundBackground

Mantle cell lymphoma (MCL)Mantle cell lymphoma (MCL) 2.8-6% of all NHLs2.8-6% of all NHLs aggressive , inevitable relapse, and poor aggressive , inevitable relapse, and poor

prognosis ( median OS of 3-4y)prognosis ( median OS of 3-4y) t(11;14) (q13;q32) resulting in cyclin D1 ot(11;14) (q13;q32) resulting in cyclin D1 o

verexpression and cell-cycle dysregulatioverexpression and cell-cycle dysregulationn

Incidence of stage Ⅳ MCL ↑ over the last Incidence of stage Ⅳ MCL ↑ over the last decade decade

No standard therapyNo standard therapy

Lenalidomide (CC-5013, RevlimidLenalidomide (CC-5013, Revlimid , , LEN)LEN) the second-generation immunomodulatory drthe second-generation immunomodulatory dr

ugsugs more potent biologic activity and less toxicity more potent biologic activity and less toxicity

than thalidomide than thalidomide Approved by FDA for the treatment of Approved by FDA for the treatment of MDSMDS ass ass

ociated with a del 5q cytogenetic abnormality, ociated with a del 5q cytogenetic abnormality, in combination with dexamethasone for the trin combination with dexamethasone for the treatment of eatment of previously treated MM patients

mechanism of action: anti-proliferative, anti-angiogenic and immunomodulatory activities

Effects of LEN on tumorEffects of LEN on tumorcells and their microenvironmentcells and their microenvironment

Lenalidomide can inhibit certain cancer cell growth; alter tumor cell microenvironment from anti-apoptotic to pro-apoptotic--- Chanan-Khan & Cheson, J Clin Oncol. 2008

Lenalidomide for NHL theraLenalidomide for NHL therapypy

There is little data to suggest direct anti-proliferative effects on NHL cells and no data assessing the potential for combination with Dex

Encouraging early results suggest that oral lenalidomide mono-therapy has a potential for clinical efficacy in NHL with manageable hematological side effects: Witzig, et al, Tuscano, et al, Lossos, et al, Wiernik, et al, Czuczman, et al, Vose, et al, Wang, et al, 2007ASH …

Objective and DesignObjective and DesignLEN+DEXLEN+DEX

In vitroIn vitro In vivoIn vivo

MCL cell line & patient samplesMCL cell line & patient samples

Growth inhibitionGrowth inhibitionapoptosisapoptosisMechanisms of actionMechanisms of action

3H-thymidine 3H-thymidine incorporation incorporation

assay, cell cycle assay, cell cycle analysisanalysis

Annexin V/PIAnnexin V/PIWestern BlotWestern Blot

MCL-SCID mouse modelMCL-SCID mouse model

TreatmentTreatmentgroupgroup

ControlControlgroupgroup

Tumor sizeTumor sizeSurvival curveSurvival curveIC50, IC50,

cell cycle arrestcell cycle arrest

time and dosetime and dose selectionselectionCaspase-dependent/Caspase-dependent/

independentindependent

Materials and methodsMaterials and methods

Cell line:Cell line: Mino cultured in 1640 Mino cultured in 1640

Patient samples:Patient samples: bone marrow and peripheral bl bone marrow and peripheral blood from 3 patients with relapsed or refractory ood from 3 patients with relapsed or refractory MCL (CD19+/CD5+ MCL (CD19+/CD5+ >95%) in IMDM>95%) in IMDM

LEN:LEN: dissolved in DMSO, 400mM stock solution dissolved in DMSO, 400mM stock solution

DEX:DEX: solubilized in ethanol, 1mM stock solution solubilized in ethanol, 1mM stock solution

Cell growth assaysCell growth assays – – 3H-thymidine incorporation3H-thymidine incorporation 1μCi/well during the last 16 hours of 72-hour cultures1μCi/well during the last 16 hours of 72-hour cultures

Cell cycle analysisCell cycle analysis – – PI staining by flow cytometryPI staining by flow cytometryApoptosis assays Apoptosis assays –– Annexin V-binding assayAnnexin V-binding assayWestern blot analysisWestern blot analysis – – Immunoblotted with Abs Immunoblotted with Abs

against caspase pathway signals and bcl-2 familyagainst caspase pathway signals and bcl-2 familyIn vivo xenograft modelsIn vivo xenograft models –– 6- to 8-week-old male CB-17 SCID mice, 1×107 Mino cells, 6- to 8-week-old male CB-17 SCID mice, 1×107 Mino cells,

palpable tumors (≥3 mm in diameter), four groups of 10 mipalpable tumors (≥3 mm in diameter), four groups of 10 mice each , LEN (50 mg kg-1 day-1) on days 1-21, DEX (0.5 mg kce each , LEN (50 mg kg-1 day-1) on days 1-21, DEX (0.5 mg kg-1 day-1) on days 1-4, 9-12, 17-20g-1 day-1) on days 1-4, 9-12, 17-20

Statistical analysisStatistical analysis – – t test ; Kaplan-Meier methodt test ; Kaplan-Meier method

ResultsResults

0

20

40

60

80

100

120

0 0.001 0.01 0.1 1 10 100

Lenalidomide (μM)

Cel

l gro

wth

(% C

on

tro

l)

Mino

PT1

PT2

PT30

20

40

60

80

100

120

0 0.001 0.01 0.1 1 10

Dexamethasone (μM)

Cel

l gro

wth

(% C

ont

rol)

Mino

PT1

PT2

PT3

0

20

40

60

80

100

120

Mino PT1 PT2 PT3

Ce

ll g

row

th (

% C

on

tro

l)

Control

LEN

DEX

LEN+DEX

A B

C

Figure 1 Lenalidomide (A) and dexamethasone (B) inhibited the growth of MCL

cells and displayed synergistic effects (C)

37.1 36.2 36.9 35.1 37.2 35.1

43.3 43.2 43.4 42.7 42.5 44.4

19.6 20.6 19.6 22.2 20.3 20.4

0

20

40

60

80

100

120

0 0.01 0.1 1 10 100

Lenalidomide (μM)

Cel

ls (%

)

PI-staining of DNA

A B

C

Figure 2 Dexamethasone induced G0/G1 arrest (B), Lenalidomide (A) had no effect on cell cycle distribution and enhanced dexamethasone-induced cell cycle arrest(C)

Control LEN DEX LEN+DEX

M1M1

M1M1

M2 M2 M2 M2

M3 M3M3 M3

M4 M4M4 M4

38.3 37.054.9

65.5

26.934.9

48.745.7

16.0 14.2 10.2 7.7

0

20

40

60

80

100

120

Control LEN DEX LEN+DEXC

ells

(%)

G0/G1 S G2/M

38.3 45.3 54.9 57.5

46.1 40.834.9 32.7

15.6 13.9 10.2 9.8

0

20

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60

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100

120

0 0.01 0.1 1

Dexamethasone (μM)

Cel

ls (%

)

B

D

0

10

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30

40

0 0.1 1 10 100

Lenalidomide (μM)

Ap

op

totic

cel

ls (%

)d2

d3

d4

d5

d6

d7

0

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0 0.01 0.1 1

Dexamethasone (μM)

Ap

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totic

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ls (%

)

d2

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d4

d5

d6

d7

PI

Annexin V

d0 d3 d4 d5 d6 d7

A B

C

Figure 3 Lenalidomide and dexamethasone induced apoptosis of MCL cells

LEN 100µM

DEX 1µM

50.3

10.0 13.7 16.7 24.9 28.1 32.8

11.8 15.6 23.0

33.4 48.2M1 M1 M1 M1 M1

M1

M1

M1 M1 M1 M1 M1

50.3

01020304050607080

0 0.1 1 10 100

Ap

op

toti

c c

ells

(%

)d2

d3

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d5

d6

d70

1020304050607080

PT1 PT2 PT3

Apo

ptot

ic c

ells

(%) Control

LEN

DEX

LEN+DEX

Figure 3 Lenalidomide plus dexamethasone synergistically induced apoptosis of MCL

cells

Control LEN DEX LEN+DEX

D

F

E

Lenalidomide (µM) with Dexamethasone

Annexin V

PI

Mino

PT1

68.4M1 M1 M1 M1

11.6 19.3 30.8

M1 M1 M1 M1

68.4

65.748.328.614.7

LEN(µM)

0 0.01 0.1 1 10 100

A B

Cleaved PARP

β-actin

Figure 4 Lenalidomide synergized with dexamethasone to trigger apoptosis of MCL cells by activating caspase 3,9 and PARP and upregulating the expression of pBcl-2, Bax and Bad

- + - +

- - + +

CLEN

DEX

- + - +

- - + +

Cleaved PARP

β-actinPT1

Mino

Cleaved cas-8

Cleaved cas-9

Cleaved cas-3

Cleaved PARP

DEX(µM)

0 0.01 0.1 1

β-actin

Cleaved PARP

LEN

DEXpBcl-2

Bcl-2

Bcl-XL

Bax

β-actin

Bad

0

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0 1 2 3 4 5 6

Weeks from treatment

Tum

or V

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e (m

m3 ) Control

LEN

DEX

LEN+DEX

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0 2 4 6 8

Weeks from treatment

% S

urvi

val

Control

LEN

DEX

LEN+DEX

Figure 5 LEN plus DEX inhibited the growth and prolonged the survival of MCL cells in vivo. LEN (50mg/kg/day) was intraperitoneally injected on days 1-21 and DEX (0.5mg/kg/day) was subcutaneously injected on days 1-4, 9-12, 17-20 after tumor burden palpable

A B

DiscussionDiscussion

LEN (25mg/d) was administered in patients LEN (25mg/d) was administered in patients with relapsed or refractory aggressive NHL uwith relapsed or refractory aggressive NHL until PD or intolerance. An objective RR of ntil PD or intolerance. An objective RR of 3535%% was observed in 49 treated patients. Respo was observed in 49 treated patients. Responses were observed in all histologic subtypes nses were observed in all histologic subtypes including DLBCL, follicular center lymphomincluding DLBCL, follicular center lymphoma, a, MCLMCL, and transformed lymphomas, and transformed lymphomas (J Clin Oncol,(J Clin Oncol, 2008) 2008)

Damaj et al reported that a multiply relapsed,Damaj et al reported that a multiply relapsed, heavily pretreated MCL patient with drug re heavily pretreated MCL patient with drug resistance to thalidomide re-achieved a PR aftesistance to thalidomide re-achieved a PR after treatment with the combination of thalidor treatment with the combination of thalidomide and DEX.mide and DEX. (Leukemia, 2003)(Leukemia, 2003)

In vitro LEN alone induced a slight growth inhibition and apoptosis o

f MCL cells. LEN plus DEX significantly increased the cytotoxic effects on

MCL cells in a dose- and time-dependent fashion. MCL patient cells displayed a stronger apoptotic response to

LEN/DEX than Mino cells

In vivo LEN/DEX synergistically delayed tumor growth and prolonge

d the survival of tumor-bearing mice, indicating that a combin

ation of LEN and DEX may be a promising regimen for the tr

eatment of MCL in vivo.

Cell cycle arrestCell cycle arrestIn this study In this study LEN had no effect on cell cycle distribution in Mino cellLEN had no effect on cell cycle distribution in Mino cell

s. s. DEX arrested cell cycle progression in G0/G1DEX arrested cell cycle progression in G0/G1 addition of LEN significantly enhanced the cell number addition of LEN significantly enhanced the cell number

of cells accumulating in the G0/G1 phase of cells accumulating in the G0/G1 phase

Previous studiesPrevious studies LEN alone induced G0/G1 growth arrest in MM cells anLEN alone induced G0/G1 growth arrest in MM cells an

d Namalwa cells, leading to a significant growth inhibitd Namalwa cells, leading to a significant growth inhibitionion

LEN arrested Namalwa cells in G0/G1, while it didn’t aLEN arrested Namalwa cells in G0/G1, while it didn’t affect the cell cycle progression of Jeko-1 cells. However,ffect the cell cycle progression of Jeko-1 cells. However, LEN synergized with DEX to induce G0/G1 arrest result LEN synergized with DEX to induce G0/G1 arrest resulting in significant inhibitory effect in both tumor cells .ing in significant inhibitory effect in both tumor cells .

Mechanism of apoptosisMechanism of apoptosis

Previous studiesPrevious studies the additive cytotoxicity of LEN plus DEX in MM celthe additive cytotoxicity of LEN plus DEX in MM cel

ls was associated with dual activation of DEX-inducls was associated with dual activation of DEX-induced caspase-9 and LEN-mediated caspase-8 cascadesed caspase-9 and LEN-mediated caspase-8 cascades

In this studyIn this study single-agent LEN and DEX activated both caspase-8 single-agent LEN and DEX activated both caspase-8

and caspase-9. LEN slightly activated these caspaseand caspase-9. LEN slightly activated these caspases, but had significantly enhanced DEX-induced actis, but had significantly enhanced DEX-induced activation of caspase-9 and -3 and cleavage of PARP, wivation of caspase-9 and -3 and cleavage of PARP, with no apparent effect on caspase-8.th no apparent effect on caspase-8. The increase in The increase in proapoptotic Bcl-2 family members further confirproapoptotic Bcl-2 family members further confirmed that synergism in the induction of apoptosis wmed that synergism in the induction of apoptosis was mainly through mitochondrial pathwaysas mainly through mitochondrial pathways

ConclusionsConclusions LEN synergized with DEX to induce growth LEN synergized with DEX to induce growth

inhibition and apoptosis of MCL cells. inhibition and apoptosis of MCL cells. Cell cycle analysis showed that LEN was able Cell cycle analysis showed that LEN was able

to enhance DEX-induced G0/G1 arrest to enhance DEX-induced G0/G1 arrest despite no effect using LEN alone. despite no effect using LEN alone.

Synergism in the induction of apoptosis was Synergism in the induction of apoptosis was revealed mainly through mitochondrial revealed mainly through mitochondrial pathways. This synergism was more pathways. This synergism was more significant in primary MCL cells than significant in primary MCL cells than established MCL cells. established MCL cells.

this drug combination synergized to delay this drug combination synergized to delay tumor growth and improve survival of MCL-tumor growth and improve survival of MCL-bearing mice. bearing mice.