lenalidomide synergizes with dexamethasone to induce growth arrest and apoptosis of mantle cell...
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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
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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
40
60
80
100
120
0 0.01 0.1 1
Dexamethasone (μM)
Cel
ls (%
)
B
D
0
10
20
30
40
0 0.1 1 10 100
Lenalidomide (μM)
Ap
op
totic
cel
ls (%
)d2
d3
d4
d5
d6
d7
0
10
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0 0.01 0.1 1
Dexamethasone (μM)
Ap
op
totic
cel
ls (%
)
d2
d3
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
d4
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
1000
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0 1 2 3 4 5 6
Weeks from treatment
Tum
or V
olum
e (m
m3 ) Control
LEN
DEX
LEN+DEX
0
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120
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.